CN104988115A - Application of quinoline or isoquinoline small-molecular compound in culture and cryopreservation of multipotential stem cells of human - Google Patents
Application of quinoline or isoquinoline small-molecular compound in culture and cryopreservation of multipotential stem cells of human Download PDFInfo
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Abstract
The invention relates to an application of a quinoline or isoquinoline small-molecular compound in culture and cryopreservation of multipotential stem cells of human. The application is characterized in that after dissociation and cryopreservation of the multipotential stem cells of human, the quinoline or isoquinoline small-molecular compound with proper concentration is applied, so that survival and clonal formation of the multipotential stem cells of human can be obviously promoted, and the undifferentiated state and the pluripotency of the multipotential stem cells can be maintained. The method is applicable to the culture mode of a feeder layer and a feeder-free layer of the multipotential stem cells of human. The method is simple and reliable, low in cost and stable and high-efficiency.
Description
Technical field
The present invention relates to quinoline or iloquinoline derivative micromolecular compound to cultivate and application in freezen protective and implementation method at human pluripotent stem cells.
Background technology
Embryonic stem cell (ESCs) derives from the inner cell mass of embryonic blastula, has unlimited multiplication capacity and multi-lineage potential.1998, the Tomson of Wisconsin university of the U.S. etc. (Tomson et al., 1998) were separated from blastaea inner cell mass manually in vitro fertilization first and obtain human embryo stem cell, and successfully carried out vitro culture and build and be.Because it can Immortalization in vitro, and the various cell types in three germinal layer sources can be divided into, old friend's embryonic stem cell becomes regenerative medicine, organizational project, drug screening, developmental biology grind the field such as high grinds high focus (Ohgushi and Sasai, 2011).Set up due to human embryo stem cell and relate to the problem such as ethics and law, it grinds and to be highly very limited with application.Japanese scholars ShinyaYamanaka was at 2006 and 2007, by importing four transcription factors (Oct4, Sox2, Klf4 and c-Myc), be induced multi-potent stem cells (Takahashi andYamanaka, 2006 by the skin flbroblast reprogrammed of mouse and people respectively first; Takahashi et al., 2007), and therefore contribution obtains the Nobel prize's soul of 2012.After this, differently grind high group and use the method for modification to obtain induced multi-potent stem cells respectively.Induced multi-potent stem cells and embryonic stem cell are all multipotential stem cell, there is the external unlimited amplification identical with embryonic stem cell and differentiation capability, and there is not the problem of ethics and law etc., therefore have broad application prospects and huge economic worth at regenerative medicine and field of tissue engineering technology.On the other hand, can obtain induced multi-potent stem cells as required from the cell in patient source, this is that individualized treatment in the future brings hope.
The initial culture condition of human pluripotent stem cells is by growing in the cell culture vessel of the substrate bag quilts such as gelatin, and by feeder layer cells supports such as rat embryo fibroblast cell or other inoblasts, also need to knock out serum replacement as culture medium mainly additive simultaneously.Cultural method containing feeder layer is relatively loaded down with trivial details, expensive, and containing animal source composition in growing environment, likely cause immunological rejection or introduce virus, this grinds high and clinical application to seriously limiting basis.Grind high persons to start to inquire into exploitation without the human pluripotent stem cells cultural method of feeder layer, the report of the method for cultivating without feeder layer about human pluripotent stem cells is more and more.Comprise cell adhesion molecule, the matrigel as ln, vitronectin and fibronectin etc. is proved to be the survival and Clone formation that can improve without human pluripotent stem cells under feeder layer culture condition.Matrigel is the matrigel extracted from the EHS mouse tumor being rich in extracellular matrix protein, at present human pluripotent stem cells grind high in be widely used.Without feeder layer cells training method to human pluripotent stem cells grind high and application bring great convenience, the training method of feeder layer will be replaced gradually.But relative to the training method containing feeder layer, obtain human pluripotent stem cells by the training method without feeder layer and want difficulty many, the survival rate of cell and cloning efficiency are very low.Therefore, culture condition without feeder layer is improved to promote that human pluripotent stem cells survival and Clone formation are that of future is important and grind high direction.
In order to realize the application of multipotential stem cell, amplification in vitro must be carried out to obtain a large amount of cell sources to it, and realize high-efficiency freezing preservation.But very easily there is a large amount of necrocytosiss in human pluripotent stem cells, this becomes its amplification in vitro, huge obstacle that is high and clinical application is ground on basis in cultivation operating process.The step such as cell expansion ex vivo and process of cryopreservation relate to cell dissociation, cryoprotectant immigration, Ultra-cryofreezing preservation, rapid fluid resuscitation, cryoprotectant shift out.But, be dissociated into single celled human pluripotent stem cells and a large amount of apoptosis (Ohgushi et al., 2010) very easily occur, so that cannot subsequent operations be carried out.In addition, although the cytolemma of human embryo stem cell can maintain its integrity after very low temperature slow freezing, the function of cell suffers damage, and a large amount of cell is at resuscitation process generation apoptosis, thus cause extremely low anabiosis rate (Heng et al., 2006).For these problems; current lot of domestic and international grinds that high person is just attempting by changing cell dissociation method, improves cryogenic freezing method, screening Novel freezing protective material and grind the aspects such as high apoptotic mechanism find stable, reliably human pluripotent stem cells cultivate and Techniques of preserving, grind high and clinical application for science and provide safeguard.Although these work make some progress, the cultivation of the raising human pluripotent stem cells surviving rate that still neither one is simple and reliable, cheap so far, repeatability is strong and freezing and storing method.
Summary of the invention
The invention provides quinoline or iloquinoline derivative micromolecular compound to cultivate and application in freezen protective and implementation method at human pluripotent stem cells, significantly can promote that human pluripotent stem cells is survived and Clone formation, and keep undifferentiated state and the versatility of multipotential stem cell.
People's induced multi-potent stem cells that in the inventive method, human pluripotent stem cells comprises human embryo stem cell or utilizes animal nutrition to obtain.
Human pluripotent stem cells survival rate when being dissociated into unicellular going down to posterity is extremely low, one of feature of the present invention is by the quinoline or iloquinoline derivative micromolecular compound that add proper concn in the substratum of front and back of dissociating, and can significantly improve the survival and Clone formation that are dissociated into single celled human pluripotent stem cells.Its concentration can the scope of 1-100 μM.
Human pluripotent stem cells carries out frozen with unicellular form, viability rate is extremely low, one of feature of the present invention is quinoline by adding proper concn in frozen storing liquid, resuscitation fluid or substratum or iloquinoline derivative micromolecular compound, can significantly improve human pluripotent stem cells survival and the Clone formation of freezen protective.Its concentration can the scope of 1-100 μM.
The inventive method be applicable to human pluripotent stem cells containing feeder layer and the training method without feeder layer cells.
For the training method containing feeder layer cells, feeder layer cells growth is in the cell culture vessel of matrigel bag quilt, and matrigel can be the matrigel of gelatin or other any support feeder layer cells growth.Feeder layer cells is can for the cell type not possessing any backer's multipotential stem cell growth of splitting ability adopting the chemical treatments such as irradiation or ametycin to cross.
For the training method without feeder layer cells, cell culture vessel needs with matrigel bag quilt, and matrigel can be commercialization or the self-control matrigel of Matrigel or other any backer's multipotential stem cell growth.
The inventive method is except the human pluripotent stem cells being applicable to unicellular form, and for the human pluripotent stem cells being dissociated into minicell lump form, after cultivation or cryopreservation resuscitation, quinoline or iloquinoline derivative micromolecular compound have similar promoter action.Dissociating the solution of human pluripotent stem cells can for pancreatin, TrypLE, Dispase, collagenase or EDTA etc.
The invention provides a kind of human pluripotent stem cells frozen storing liquid, utilize the frozen human pluripotent stem cells of this frozen storing liquid to have good survival rate and Clone formation efficiency.This frozen storing liquid for adding the business-like frozen storing liquid of proper concn quinoline or iloquinoline derivative micromolecular compound, or can make frozen storing liquid by oneself.
The invention provides a kind of human pluripotent stem cells resuscitation fluid, the human pluripotent stem cells after utilizing this resuscitation fluid to recover has good survival rate and Clone formation efficiency.This resuscitation fluid can for adding the basic mediums such as DMEM, DMEM/F12, KnockoutDMEM or Knockout DMEM/F12 of proper concn quinoline or iloquinoline derivative micromolecular compound, or with the commercialization of backer's multipotential stem cell growth of this several basic medium preparation or self-made medium, or other commercialization or self-control resuscitation fluid.
The inventive method is applicable to slow cryopreservation method and the glass frozen preservation method of human pluripotent stem cells.For slow cryopreservation method, relate to and utilize programmed temperature control instrument to carry out frozen method with the frozen speed of 1-10 DEG C/min, or utilize the cryopreservation methods of business-like freezing storing box, or other conventional gradient cooling freezing preservation method; For glass frozen preservation method, relate to the glass frozen preservation method utilizing straw or other modifying device.
The inventive method is by human pluripotent stem cells cultivation or process of cryopreservation, the quinoline of simple interpolation proper concn or iloquinoline derivative micromolecular compound can significantly improve and dissociate or cell survival after Cryopreservation and Clone formation, and still maintain undifferentiated state and the versatility of human pluripotent stem cells.The method operation is very easy, and cheap, successful, can stablize and obtain a large amount of cells, grind high and demand that is clinical application with satisfied basis, have a extensive future.
Accompanying drawing explanation
Fig. 1 is the human embryo stem cell Clone formation and the form thereof that contain feeder layer cultivation
Fig. 2 be without feeder layer cultivate human embryo stem cell Clone formation and form
Fig. 3 is human embryo stem cell cell survival when being dissociated into single cell culture
Fig. 4 is with the frozen rear comparative survival rate of cells of human embryo stem cell recovery of unicellular form
Fig. 5 is the undifferentiated state qualification of human embryo stem cell
Fig. 6 is the differentiation potential qualification of human embryo stem cell
Embodiment
Be example with the H9 cell of human embryo stem cell in following embodiment, and to cultivate conventional reagent with human pluripotent stem cells be in conventional manner that the present invention is described in detail.
Experiment material
L-glutaminate, Basic fibroblast growth factor (bFGF), ametycin (MMC), gelatin, Matrigel, TrypLE, collagenase IV, dimethyl sulfoxide (DMSO) (DMSO), basic medium DMEM and DMEM/F12.
The formation of human pluripotent stem cells perfect medium: add 20% (V/V) serum substitute KSR, 2mM glutamine, the nonessential amino acid of 0.1mM, 4ng/ml recombinant human bfgf and 0.055mM beta-mercaptoethanol in DMEM/F12.
Human pluripotent stem cells is without feeder layer substratum: buy commercialization substratum Essential 8.
Feeder layer is the mouse embryo fibroblasts (MEF) of irradiation or ametycin process, and its substratum is formed: add 10% (V/V) FBS, 2mM glutamine and the nonessential amino acid of 0.1mM in DMEM.
Embodiment one
Human pluripotent stem cells containing feeder layer cells is cultivated
1) by the Mouse feeder confluent monolayer cells MEF of irradiation or ametycin process according to 2-3x10
5individual cell/cm
2density be seeded in the culture dish of 0.1% gelatin bag quilt.
2) by human pluripotent stem cells DPBS rinsing one time, add appropriate dissociation solution (as collagenase IV, TrypLE, EDTA, Dispase or pancreatin etc.), for collagenase IV (1mg/ml), add isopyknic substratum after digestion certain hour and stop digestion reaction and blow and beat clone is disperseed.
3) the standing or centrifugal 5min collecting cell of 1000rpm, adds appropriate substratum re-suspended cell, is added with quinoline or the iloquinoline derivative micromolecular compound (final concentration 1-100 μM) of proper concn in substratum after removing supernatant.
4) cell suspension is divided in 48 well culture plates being covered with feeder layer cells, be placed in 37 DEG C, 5%CO
2cultivate in incubator.Next day gets final product replaced medium.During cellar culture, every day replaced medium.
The substratum that re-suspended cell is used can be the commercialization of any backer's multipotential stem cell growth or self-made medium, the present embodiment adopts human pluripotent stem cells substratum conventional at present, consists of in DMEM/F12 and adds 20% (V/V) serum substitute KSR, 2mM glutamine, the nonessential amino acid of 0.1mM, 4ng/ml recombinant human bfgf and 0.055mM beta-mercaptoethanol.
The invention provides a kind of human pluripotent stem cells substratum, for the resuspended of cell after dissociating with cultivate, consist of the quinoline or the iloquinoline derivative micromolecular compound (final concentration 1-100 μM) that add proper concn in the commercialization of any backer's multipotential stem cell growth or self-made medium.The present embodiment adopts the quinoline or the iloquinoline derivative micromolecular compound (final concentration 4 μMs) that add proper concn in above-mentioned conventional human pluripotent stem cells substratum.
Result presents for human embryo stem cell H9 herein, and chooses two kinds of quinoline or iloquinoline derivative micromolecular compound carries out accompanying drawing explanation.Dissociate and postvaccinal human embryo stem cell, substratum significantly increases the quantity (clone of meter alkaline phosphatase AP stained positive) (accompanying drawing 1A) of Clone formation containing N-[2-[P-bromobenzene propylene alkali is amino] ethyl]-5-isoquinoline sulfonaide (being called for short H89 again) and cinchonine; Their form (accompanying drawing 1B) adding not change human embryo stem cell.
Embodiment two
Human pluripotent stem cells without feeder layer cells is cultivated
1) add appropriate Matrigel coating buffer in Tissue Culture Dish, 4 DEG C bag spent the night or 37 DEG C bag by more than 2h.
2) by human pluripotent stem cells DPBS rinsing one time, add appropriate dissociation solution (as collagenase IV, TrypLE, EDTA, Dispase or pancreatin etc.), for TrypLE, add after digestion certain hour isopyknic substratum stop digestion reaction and piping and druming to make it be dispersed into unicellular.
3) the centrifugal 5min collecting cell of 1000rpm, adds appropriate substratum re-suspended cell after removing supernatant, is added with quinoline or the iloquinoline derivative micromolecular compound (final concentration 1-100 μM) of proper concn in substratum.
4) cell suspension is divided in 48 well culture plates being covered with feeder layer cells, be placed in 37 DEG C, 5%CO
2cultivate in incubator.Next day gets final product replaced medium.During cellar culture, every day replaced medium.
The substratum used of re-suspended cell can for the commercialization of any backer's multipotential stem cell growth or self-control be without feeder layer substratum, the present embodiment adopt commonly use at present without feeder layer human pluripotent stem cells substratum Essential 8.
The invention provides a kind of human pluripotent stem cells without feeder layer substratum, for the resuspended of cell after dissociating with cultivate, consist of the quinoline or the iloquinoline derivative micromolecular compound (final concentration 1-100 μM) that add proper concn in the commercialization or self-made medium that any backer's multipotential stem cell grows under without feeder layer condition.The present embodiment adopts the quinoline or the iloquinoline derivative micromolecular compound (final concentration 4 μMs) that add proper concn in Essential 8 substratum.
Result presents for human embryo stem cell H9 herein, and chooses two kinds of quinoline or iloquinoline derivative micromolecular compound carries out accompanying drawing explanation.To be dissociated into single celled human embryo stem cell is seeded in culture plate, adds N-[2-[P-bromobenzene propylene alkali amino] ethyl in substratum]-5-isoquinoline sulfonaide (being called for short H89 again) and cinchonine significantly increase Clone formation (accompanying drawing 1A); Their form (accompanying drawing 1B) adding not change human embryo stem cell.
Embodiment three
The impact that quinoline or iloquinoline derivative micromolecular compound are survived on human pluripotent stem cells
In order to verify whether quinoline or iloquinoline derivative micromolecular compound decrease necrocytosis, in the present embodiment, be seeded in the orifice plate of Matrigel bag quilt by being dissociated into single celled human embryo stem cell, with the mode cellar culture 8h without feeder layer cells, then collecting cell, add 4 μMs of PI staining fluids to dye to dead cell, and pass through flow cytometry analysis.Result adds as Fig. 3 A, N-[2-[P-bromobenzene propylene alkali is amino] ethyl]-5-isoquinoline sulfonaide (being called for short H89 again) the quantity considerably reducing dead cell.
In addition, we utilize MTT have detected to dissociate postvaccinal human embryo stem cell growing state.As shown in Figure 3 B, N-[2-[P-bromobenzene propylene alkali amino] ethyl]-5-isoquinoline sulfonaide add the survival rate significantly improving human embryo stem cell.
Embodiment four
The freezen protective of human pluripotent stem cells
1), before human pluripotent stem cells freezen protective, add quinoline or the iloquinoline derivative micromolecular compound (final concentration 1-100 μM) of proper concn in substratum, hatch for 37 DEG C.In the present embodiment, with 3 μMs of N-[2-[P-bromobenzene propylene alkali amino] ethyl]-5-isoquinoline sulfonaide (being called for short H89 again), hatching 2h (being not limited only to 2h) for example is described.
2) dissociate after removing substratum, the cell of collection adds appropriate substratum re-suspended cell.
3) isopyknic human pluripotent stem cells frozen storing liquid (being wherein added with quinoline or iloquinoline derivative micromolecular compound) is added, its final concentration is made to be 1-100 μM (in the present embodiment, for 3 μMs of N-[2-[P-bromobenzene propylene alkali is amino] ethyl]-5-isoquinoline sulfonaide).
4) gently mixing after, cell suspension is divided in cryopreservation tube, is then placed in the freezing storing box of 4 DEG C of precoolings, be put into-80 DEG C spend the night after be transferred to Liquid nitrogen storage.
Or the cell after collected by centrifugation, directly adding human pluripotent stem cells frozen storing liquid (wherein containing 1-100 μM of quinoline or iloquinoline derivative micromolecular compound) carries out resuspended, cell suspension is divided in cryopreservation tube, then be placed in the freezing storing box of 4 DEG C of precoolings, be put into-80 DEG C spend the night after be transferred to Liquid nitrogen storage.
The invention provides several human pluripotent stem cells frozen storing liquid, it is characterized in that the quinoline or the iloquinoline derivative micromolecular compound (during use, final concentration is 1-100 μM) that wherein add proper concn:
A. human pluripotent stem cells perfect medium: DMSO: KSR (or FBS) (V/V)=1: 1: 3, uses with human pluripotent stem cells perfect medium 1: 1 ratio, wherein preferably uses KSR.
B. human pluripotent stem cells perfect medium: DMSO (V/V)=4: 1, uses with human pluripotent stem cells perfect medium 1: 1 ratio.
C.DMSO: KSR (or FBS) (V/V)=1: 4, uses with human pluripotent stem cells perfect medium 1: 1 ratio, wherein preferably uses KSR.
Several human pluripotent stem cells frozen storing liquid is containing 20%DMSO above, and also the frozen storing liquid that can be formulated as containing 10%DMSO directly uses, and does not need to use with human pluripotent stem cells perfect medium 1: 1 ratio.The present invention, except providing above-mentioned several preferred frozen storing liquid, also comprises other formula based on this several frozen storing liquid, and its another feature is that the working concentration of DMSO can be 5%-20%.
In addition, the human pluripotent stem cells substratum in frozen storing liquid can use the basic mediums such as DMEM, DMEM/F12, Knockout DMEM or KnockoutDMEM/F12 to substitute.Preferred, frozen storing liquid used is human pluripotent stem cells perfect medium in the present embodiment: the formation of DMSO: KSR (V/V)=1: 1: 3.
For the use of quinoline or iloquinoline derivative micromolecular compound, a kind of scheme: the quinoline or the iloquinoline derivative micromolecular compound that add proper concn in the medium, use with frozen storing liquid 1: 1, make the final concentration of quinoline or iloquinoline derivative micromolecular compound at 1-100 μM; Another kind of scheme: the quinoline or the iloquinoline derivative micromolecular compound that add proper concn in frozen storing liquid, during freeze-stored cell, the final concentration of quinoline or iloquinoline derivative micromolecular compound is at 1-100 μM.
Except DMSO, during with other cryoprotectant freezen protective human pluripotent stem cells, add quinoline or iloquinoline derivative micromolecular compound in frozen storing liquid and can improve human pluripotent stem cells survival and Clone formation equally.
In the present embodiment, we adopt AlamarBlue to detect Cryopreservation human embryo stem cell survival situation (relative Reduced% is used for representing comparative survival rate of cells).Compared with control group, N-[2-[P-bromobenzene propylene alkali is amino] ethyl]-5-isoquinoline sulfonaide (being called for short H89 again) pretreated experimental group before only dissociating, after frozen one-cell human embryos stem cell recovery, comparative survival rate of cells significantly improves (accompanying drawing 4A); Only add H89 in frozen storing liquid and significantly increase comparative survival rate of cells (accompanying drawing 4B) equally; H89 pre-treatment before dissociating also adds H89 in frozen storing liquid, further increases comparative survival rate of cells (accompanying drawing 4C).
Embodiment five
The recovery of human pluripotent stem cells
1) frozen human pluripotent stem cells is 37
rapid fluid resuscitation in bath.
2) slowly drip appropriate basic medium or human pluripotent stem cells substratum or resuscitation fluid in cell suspension, dropping limit, limit slowly mixes.
3) the centrifugal 5min of 1000rpm, adds appropriate resuscitation fluid re-suspended cell after removing supernatant, be added with quinoline or the iloquinoline derivative micromolecular compound (final concentration 1-100 μM) of proper concn in resuscitation fluid.
4) culture dish to Matrigel bag quilt is divided by cell suspension to cultivate.Next day gets final product replaced medium.During cellar culture, every day replaced medium.
The invention provides a kind of human pluripotent stem cells resuscitation fluid, this resuscitation fluid can for adding the basic mediums such as DMEM, DMEM/F12, Knockout DMEM or Knockout DMEM/F12 of proper concn quinoline or iloquinoline derivative micromolecular compound (final concentration 1-100 μM), or with the commercialization of backer's multipotential stem cell growth of this several basic medium preparation or self-made medium, or other commercialization or self-control resuscitation fluid.Resuscitation fluid can use in 0-24h after human pluripotent stem cells recovery.Preferred, in the present embodiment, resuscitation fluid is configured to add quinoline or iloquinoline derivative micromolecular compound in human pluripotent stem cells substratum, and wherein substratum can be the substratum of any backer's multipotential stem cell growth.
In the present embodiment, we adopt AlamarBlue to detect Cryopreservation human embryo stem cell survival situation.As shown in accompanying drawing 4D, add N-[2-[P-bromobenzene propylene alkali is amino] ethyl]-5-isoquinoline sulfonaide (being called for short H89 again) in frozen one-cell human embryos stem cell recovery wild Oryza species and significantly increase comparative survival rate of cells.Based on the result of embodiment four, preferred, add H89 in H89 pre-treatment, frozen storing liquid before dissociating, and use H89 and further increase cell survival rate (accompanying drawing 4E) after recovery.
The experimental technique used in the present invention, comprise MTT and detect cell survival, AlamarBlue detection cell survival, alkaline phosphatase staining, protein immunization fluoroscopic examination, caryogram detection (G is with analysis), external embryoid body (EB) formation and myocardial cell and neural progenitor cell differentiation, these experimental techniques are ordinary method.
Quinoline or the process of iloquinoline derivative micromolecular compound is used in human pluripotent stem cells long-term cultivation process, its colony morphology is normal (accompanying drawing 5A cultivates containing feeder layer cells and 6A cultivates without feeder layer cells) still, caryogram display normal (accompanying drawing 5B), alkaline phosphatase staining is positive (accompanying drawing 5C).Immunofluorescence test versatility protein expression is positive (accompanying drawing 5D).These cells can form EB (accompanying drawing 6B) in vitro, and Spontaneous Differentiation is the cell (accompanying drawing 6C-E) of entoderm (Endoderm), mesoderm (Mesoderm) and ectoderm (Ectoderm); Can directed differentiation be neural progenitor cell (NPCs) (accompanying drawing 6F).Immunofluorescence test shows that in the myocardial cell of directed differentiation and neural progenitor cell, corresponding labelled protein is expressed normal (accompanying drawing 6G and H).These results illustrate that the human pluripotent stem cells that life-time service quinoline or iloquinoline derivative micromolecular compound are cultivated still can keep undifferentiated state and versatility.
Claims (9)
1. quinoline or the application of iloquinoline derivative micromolecular compound in human pluripotent stem cells cultivation and freezen protective, it is characterized in that can significantly improve human pluripotent stem cells dissociates and cell survival and Clone formation after freezen protective, and keep undifferentiated state and the versatility of multipotential stem cell.
2. application according to claim 1; described quinoline or iloquinoline derivative micromolecular compound are selected from lower group: 4-fluorine isoquinoline 99.9-5-SULPHURYL CHLORIDE; 6-bromo-1; 2-dihydro-1-oxo-4-isoquinoline sulfonyl chloride; berberine hydrochloride; cinchonine; the chloro-5-isoquinoline sulfonyl chloride of 1-; HQS, 2-ethanoyl-1,2; 3; 4-tetrahydrochysene-7-isoquinoline sulfonyl chloride, N-[2-[P-bromobenzene propylene alkali is amino] ethyl]-5-isoquinoline sulfonaide and 1-[N, O-bis-(5-isoquinolinesulfonylcompounds)-N-methyl-L-type tyrosine]-4-phenylpiperazine.
3. application according to claim 1, the concentration range of quinoline or iloquinoline derivative micromolecular compound is 1-100 μM.
4. application according to claim 1, before the human pluripotent stem cells that dissociates, uses and hatches containing the quinoline of proper concn or iloquinoline derivative micromolecular compound, can significantly improve human pluripotent stem cells and dissociate and cell survival and Clone formation after Cryopreservation.
5. application according to claim 1, after human pluripotent stem cells dissociates, adds quinoline or the iloquinoline derivative micromolecular compound of proper concn in the medium, can significantly improve human pluripotent stem cells dissociate after cell survival and Clone formation.
6. application according to claim 1, frozen human pluripotent stem cells, the resuscitation fluid containing proper concn quinoline or iloquinoline derivative micromolecular compound is used during recovery, can significantly improve the rear cell survival of human pluripotent stem cells recovery and Clone formation, resuscitation fluid comprises commercialization or self-control human pluripotent stem cells resuscitation fluid.
7. application according to claim 1, can significantly improve the rear cell survival of human pluripotent stem cells recovery and Clone formation containing the quinoline or iloquinoline derivative micromolecular compound that add proper concn in the frozen storing liquid used during cryopreserved human multipotential stem cell, frozen storing liquid comprises commercialization or self-control human pluripotent stem cells frozen storing liquid.
8. application according to claim 1, relates to the arbitrary combination of the using method of quinoline or iloquinoline derivative micromolecular compound in claim 4,6 or 7, can significantly improve the rear cell survival of human pluripotent stem cells recovery and the Clone formation of freezen protective.
9. relate to the application be combined in human pluripotent stem cells cultivation and freezen protective of quinoline or iloquinoline derivative micromolecular compound.
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| WO2017197379A1 (en) * | 2016-05-13 | 2017-11-16 | Xu Han | Cryopreservation medium and method to prevent recrystallization |
| CN110868853A (en) * | 2017-04-26 | 2020-03-06 | 纪念斯隆-凯特琳癌症中心 | Ready-to-use cryopreservation of cells |
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| CN102695790A (en) * | 2009-10-29 | 2012-09-26 | 詹森生物科技公司 | Pluripotent stem cells |
| CN104662148A (en) * | 2012-09-07 | 2015-05-27 | 日本化学研究株式会社 | Culture medium for corneal endothelial cell culture containing conditioned medium of mesenchymal stem cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017197379A1 (en) * | 2016-05-13 | 2017-11-16 | Xu Han | Cryopreservation medium and method to prevent recrystallization |
| CN110868853A (en) * | 2017-04-26 | 2020-03-06 | 纪念斯隆-凯特琳癌症中心 | Ready-to-use cryopreservation of cells |
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