CN105647861A - Kit for culturing stem cells - Google Patents
Kit for culturing stem cells Download PDFInfo
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- CN105647861A CN105647861A CN201610203507.6A CN201610203507A CN105647861A CN 105647861 A CN105647861 A CN 105647861A CN 201610203507 A CN201610203507 A CN 201610203507A CN 105647861 A CN105647861 A CN 105647861A
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- 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)
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- 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
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
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Abstract
The invention discloses a kit for culturing stem cells. The kit comprises cell culture liquid A and cell culture liquid B. The cell culture liquid A is a Knockout-DMEM culture medium containing 80-120ng/ml of cell factor LIF, 80-120ng/ml of cell factor bFGF and 20-30 microgram/ml of Pedinophyllol J. The cell culture liquid B is fetal calf serum. The kit has the advantages that umbilical cord mesenchymal stem cells can still maintain high purity and good totipotency after 15 times of generation passages by the cell culture liquid; good effect can also be achieved when the cell culture liquid is used for culturing mesenchymal stem cells; the results show that the kit can increase the generation passage time of primary mesenchymal stem cells, and the mesenchymal stem cells can still keep good cell totipotency after multiple times of generation passages.
Description
Technical field
The invention belongs to stem cell and cultivate field, be specifically related to a kind of stem cell and cultivate test kit.
Background technology
Mescenchymal stem cell (mesenchymalstemcells, MSC) is the important member of stem cell line, derives from the mesoderm and ectoderm of growing early stage. MSC finds at first in bone marrow, because of its there is multi-lineage potential, the feature such as hematopoiesis support and promotion stem cell are implanted, immunoregulation and self replication and be increasingly subject to the concern of people. If mescenchymal stem cell is in vivo or under external specific inductive condition, the Various Tissues cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium can be divided into, still there is multi-lineage potential, the injuries of tissues and organs reparation that can cause for old and feeble and pathological changes as desirable seed cell after continuous passage cultivation and freezen protective.
Mescenchymal stem cell includes umbilical cord mesenchymal stem cells and mesenchymal stem cells MSCs.
Umbilical cord mesenchymal stem cells is the stem cell that a class has self renewal, propagation and multi-lineage potential, has wide material sources, is prone to collection, storage and transport, repels without allosome, avoid the plurality of advantages such as dispute of ethic. Flow cytometry analysis finds umbilical cord mesenchymal stem cells high expressed Interstitial cell mark (CD44, CD105), integrin receptor (CD29, CD49b, CD49c, CD51), does not express hemopoietic system mark (CD34, CD45) human leucocyte antigen HLA-DR and endothelial cell marker CD31. Umbilical cord mesenchymal stem cells can be divided into osteocyte, chondrocyte, hepatocyte, myocardial cell, Skeletal Muscle Cell and neuronal cell etc. in vivo and in vitro. Current human umbilical cord mesenchymal stem cells gos deep into gradually in the clinical application researchs such as tissue engineered bone, artificial blood vessel and gene therapy, and has shown that wide application prospect. At present, to the separation and Culture of umbilical cord mesenchymal stem cells, amplification but without unified standard, there is the shortcomings such as low, the original cuiture time length of purity. The amplification of various types of one-tenth fiber adult stem cells is typically in 2��3 days initially entering exponential growth, it is more or less the same, and primary cell is due to tissue-derived difference, occurs that cell clone time difference is bigger, so, the primary cell generation time is the key separating and obtaining purpose cell.
Summary of the invention
It is an object of the invention to overcome the defect of prior art, it is provided that a kind of stem cell that can improve the mescenchymal stem cell primary cell generation time cultivates test kit, so that mescenchymal stem cell still maintains cellular omnipotency after repeatedly going down to posterity.
The above-mentioned purpose of the present invention is achieved by the techniques below scheme:
A kind of stem cell cultivates test kit, including cell culture fluid A and cell culture fluid B;
Described cell culture fluid A is the Knockout-DMEM culture medium containing 80��120ng/ml cytokine LIF, 80��120ng/ml cytokine bFGF and 20��30 �� g/mlPedinophyllolJ;
Described cell culture fluid B is hyclone.
Further, described cell culture fluid A is the Knockout-DMEM culture medium containing 100ng/ml cytokine LIF, 100ng/ml cytokine bFGF and 25 �� g/mlPedinophyllolJ.
Further, described stem cell is umbilical cord mesenchymal stem cells.
Further, described stem cell is mesenchymal stem cells MSCs.
Further, described cell culture fluid A and cell culture fluid B are aseptic, and each independent packaging.
Further, described stem cell is cultivated test kit and also includes cell washing solution and cell dissociation buffer; Described cell washing solution is normal saline; Described cell dissociation buffer is CollagenaseII aqueous solution.
Further, the CollagenaseII aqueous solution of described cell dissociation buffer to be concentration be 0.2g/100ml.
Further, described normal saline is made up of sodium chloride and water, and the concentration of sodium chloride is 0.9g/100ml.
Further, described cell culture fluid A, cell culture fluid B, cell washing solution and cell dissociation buffer are aseptic, and each independent packaging.
Above-mentioned stem cell cultivates test kit application in mescenchymal stem cell is cultivated.
Advantages of the present invention:
Cultivation test kit provided by the invention can improve the mescenchymal stem cell primary cell generation time, still maintains cellular omnipotency after making mescenchymal stem cell repeatedly go down to posterity.
Detailed description of the invention
Further illustrate the essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this. Although the present invention being explained in detail with reference to preferred embodiment, it will be understood by those within the art that, it is possible to technical scheme is modified or equivalent replacement, without deviating from the spirit and scope of technical solution of the present invention.
The preparation method of the compounds of this invention PedinophyllolJ is referring to document: HighlyOxygenatedent-Pimarane-TypeDiterpenoidsfromtheChin eseLiverwortPedinophylluminterruptumandTheirAllelopathic Activities, J.Nat.Prod.2013,76,1647-1653.
Embodiment 1: the preparation of test kit
1, the preparation of component
The preparation of cell washing solution: 9gNaCl being dissolved in deionized water and is settled to 1000ml with deionized water, autoclaving is placed in 4 DEG C of refrigerators standby.
The preparation of cell culture fluid A: cytokine LIF, cytokine bFGF and compound PedinophyllolJ are dissolved in aseptic Knockout-DMEM culture medium (liquid), the concentration of cytokine LIF is 100ng/ml, the concentration of cytokine bFGF is 100ng/ml, and the concentration of compound PedinophyllolJ is 25 �� g/ml. It is placed in 4 DEG C of refrigerators standby.
Cell culture fluid B is aseptic hyclone, is placed in-20 DEG C of refrigerators standby.
Cell dissociation buffer: CollagenaseII is dissolved in deionized water and makes the concentration of CollagenaseII be 0.2g/100ml, be placed in 4 DEG C of refrigerators standby.
2, the preparation of the subpackage of component and test kit
Stem cell test kit (1 time/box) is made up of the following component of independent packaging: 1 bottle of cell washing solution (100ml/ bottle), 1 bottle of cell culture fluid A (80ml/ bottle), 1 bottle of cell culture fluid B (20ml/ bottle) and 1 bottle of cell dissociation buffer (100ml/ bottle).
Embodiment 2: the preparation of test kit, contrasts with embodiment 1, only without PedinophyllolJ in cell culture fluid A
1, the preparation of component
The preparation of cell washing solution: 9gNaCl being dissolved in deionized water and is settled to 1000ml with deionized water, autoclaving is placed in 4 DEG C of refrigerators standby.
The preparation of cell culture fluid A: cytokine LIF and cytokine bFGF is dissolved in aseptic Knockout-DMEM culture medium (liquid), the concentration of cytokine LIF is the concentration of 100ng/ml, cytokine bFGF is 100ng/ml. It is placed in 4 DEG C of refrigerators standby.
Cell culture fluid B is aseptic hyclone, is placed in-20 DEG C of refrigerators standby.
Cell dissociation buffer: CollagenaseII is dissolved in deionized water and makes the concentration of CollagenaseII be 0.2g/100ml, be placed in 4 DEG C of refrigerators standby.
2, the preparation of the subpackage of component and test kit
Stem cell test kit (1 time/box) is made up of the following component of independent packaging: 1 bottle of cell washing solution (100ml/ bottle), 1 bottle of cell culture fluid A (80ml/ bottle), 1 bottle of cell culture fluid B (20ml/ bottle) and 1 bottle of cell dissociation buffer (100ml/ bottle).
Embodiment 3: the separation of umbilical cord mesenchymal stem cells is cultivated with rapid amplifying
The test kit prepared by embodiment 1 (experimental group) and embodiment 2 (matched group) respectively carries out the separation of umbilical cord mesenchymal stem cells and cultivates with rapid amplifying.
The preparation of cell culture fluid: cell culture fluid A and cell culture fluid B are mixed according to the volume ratio of 5:1.
1, the in vitro umbilical cord of sterile collection (source is people), it is immersed in and (is made up of cell culture fluid, penicillin and streptomycin containing dual anti-cell culture fluid containing dual anti-cell culture fluid, the concentration of penicillin be 1g/100ml, streptomycin concentration be 1g/100ml) in, glass container seals transport to laboratory.
2, in Biohazard Safety Equipment, clip is about the umbilical cord of 10cm length, cleans blood stains with cell washing solution; Remove with dissecting knife outer by amniotic membrane, 2 umbilical veines and 1 umbilical artery, obtain Wal Tong Shi glue (the embryo's mucoid connective tissue between arteriovenous).
3, Wal Tong Shi glue is shredded into meat paste shape, transfer in centrifuge tube, add 20ml cell dissociation buffer, mix 37 DEG C of digested overnight (about 6 hours); Draw liquid phase, centrifugal (2500rpm, 5 minutes), collect precipitation (primary cell).
4, with the resuspended primary cell of cell culture fluid, it is inoculated in the 0.2% coated culture bottle of gelatin (every 106Individual cell is inoculated into 1 25cm2Culture bottle), it is placed in 37 DEG C, 5%CO2Incubator is cultivated; Continuous observation cell attachment growing state, cell most adherent rear (about 24 hours) is replaced by new cell culture fluid, removing non-attached cell and impurity, within later every 3 days, half amount changes the cell culture fluid (half of half amount and archeocyte culture fluid volume; Also can every 4 days half amount change cell culture fluid), primitive cell culture after 7 days cell density reach 90%.
5, primitive cell culture carries out first time and goes down to posterity after reaching 90% to cell density, all reaches 90% (cultivating 2��3 days) time at cell density afterwards and goes down to posterity;Continue to go down to posterity 20 times (after namely first time goes down to posterity, continuous passage 19 times again).
Embodiment 4: cell purity measures
The test kit that respectively prepared by Example 1 (experimental group) and embodiment 2 (matched group) carries out flow cytomery by the go down to posterity cell of 20 times of embodiment 3 method separation and Culture, to verify the purity of cell sample.
Detected the 20th time the collection of illustrative plates of the surface antigen CD29 of the cell sample after going down to posterity respectively by flow cytometer, the positive rate of experimental group cell sample is 96.6%, and the positive rate of cellular control unit sample is only 80.8%. It is shown that adopt cell culture fluid provided by the invention to be gone down to posterity by primary cell after 20 times, the purity of cell is still very high.
Embodiment 5: totipotency is verified
For confirm umbilical cord mesenchymal stem cells multi-lineage potential, the present embodiment the in embodiment 3 the 15th time is gone down to posterity after cell sample identify to the potential of osteoblast and Adipocyte Differentiation.
1, to osteoblast differentiation
By cell sample with 3000cells/cm2Inoculation, be subsequently adding Osteogenic Induction Medium (add in Knockout-DMEM culture medium dexamethasone to 100nM, ascorbic acid to 50 ��Ms, sodium ��-glycerophosphate is to 10mM), in 37 DEG C, 5%CO2Incubator is cultivated, observes after about 5��7 days.
Result experimental group can be observed following phenomenon: cell confluency becomes monolayer, and cell process interconnects, and overlap can grow and do not come in contact suppression; The cell of overlapping growth gradually forms nodositas, collagen deposition and calcium deposition subsequently, and the fine and close light tight agglomerate material of circle occurs in iuntercellular, it was shown that cell sample can to osteoblast differentiation.
Matched group does not observe experimental group finding phenomenon, illustrates to use the umbilical cord mesenchymal stem cells cultivated of test kit of embodiment 2 preparation basic forfeiture after 15 generations of going down to posterity to continue to be divided into osteoblastic ability.
2, to Adipocyte Differentiation
By cell sample with 3000cells/cm2Inoculation, be subsequently adding Adipogenic induction culture medium (add in Knockout-DMEM culture medium dexamethasone to 1 ��M, methyl-isobutyl xanthine is to 0.5mM, insulin to 10 �� g/ml, indomethacin to 100 ��Ms), continue to cultivate.
Result experimental group is following phenomenon as seen: within the 2nd day, cell confluency becomes monolayer; Within 2 weeks, rear section cellular morphology becomes round, and occurs that bright circular fat drips in endochylema; As time went on, round cell increases gradually. Show that cell sample can to Adipocyte Differentiation.
Matched group does not observe experimental group finding phenomenon, illustrates to use the umbilical cord mesenchymal stem cells cultivated of test kit of embodiment 2 preparation to go down to posterity and basic after 15 generations loses the ability continuing to be divided into adipose cell.
Result above shows, adopts cell culture fluid provided by the invention to be gone down to posterity by umbilical cord mesenchymal stem cells after 15 times, and cell still maintains significantly high purity and good totipotency.
Adopt cell culture fluid provided by the invention to cultivate mesenchymal stem cells MSCs, also achieve effect good equally.
The effect of above-described embodiment indicates that the essentiality content of the present invention, but does not limit protection scope of the present invention with this. It will be understood by those within the art that, it is possible to technical scheme is modified or equivalent replacement, without deviating from essence and the protection domain of technical solution of the present invention.
Claims (10)
1. a stem cell cultivates test kit, it is characterised in that: include cell culture fluid A and cell culture fluid B;
Described cell culture fluid A is the Knockout-DMEM culture medium containing 80��120ng/ml cytokine LIF, 80��120ng/ml cytokine bFGF and 20��30 �� g/mlPedinophyllolJ;
Described cell culture fluid B is hyclone.
2. stem cell according to claim 1 cultivates test kit, it is characterised in that: described cell culture fluid A is the Knockout-DMEM culture medium containing 100ng/ml cytokine LIF, 100ng/ml cytokine bFGF and 25 �� g/mlPedinophyllolJ.
3. stem cell according to claim 1 cultivates test kit, it is characterised in that: described stem cell is umbilical cord mesenchymal stem cells.
4. stem cell according to claim 1 cultivates test kit, it is characterised in that: described stem cell is mesenchymal stem cells MSCs.
5. stem cell according to claim 1 cultivates test kit, it is characterised in that: described cell culture fluid A and cell culture fluid B are aseptic, and each independent packaging.
6. stem cell according to claim 1 cultivates test kit, it is characterised in that: also include cell washing solution and cell dissociation buffer; Described cell washing solution is normal saline; Described cell dissociation buffer is CollagenaseII aqueous solution.
7. stem cell according to claim 6 cultivates test kit, it is characterised in that: described cell dissociation buffer is concentration is the CollagenaseII aqueous solution of 0.2g/100ml.
8. stem cell according to claim 6 cultivates test kit, it is characterised in that: described normal saline is made up of sodium chloride and water, and the concentration of sodium chloride is 0.9g/100ml.
9. stem cell according to claim 6 cultivates test kit, it is characterised in that: described cell culture fluid A, cell culture fluid B, cell washing solution and cell dissociation buffer are aseptic, and each independent packaging.
10. the arbitrary described stem cell of claim 1��9 cultivates test kit application in mescenchymal stem cell is cultivated.
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| CN201610203507.6A CN105647861A (en) | 2016-03-31 | 2016-03-31 | Kit for culturing stem cells |
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| CN201610203507.6A CN105647861A (en) | 2016-03-31 | 2016-03-31 | Kit for culturing stem cells |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965335A (en) * | 2011-09-01 | 2013-03-13 | 北京清美联创干细胞科技有限公司 | Kit for mesenchymal stem cell culture and application thereof |
| CN103087982A (en) * | 2011-10-27 | 2013-05-08 | 北京清美联创干细胞科技有限公司 | Kit and method capable of quickly separating adipose tissue-derived stem cells |
| CN105130796A (en) * | 2015-10-22 | 2015-12-09 | 云南民族大学 | Diterpenoid compound and preparing method and application thereof |
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2016
- 2016-03-31 CN CN201610203507.6A patent/CN105647861A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965335A (en) * | 2011-09-01 | 2013-03-13 | 北京清美联创干细胞科技有限公司 | Kit for mesenchymal stem cell culture and application thereof |
| CN103087982A (en) * | 2011-10-27 | 2013-05-08 | 北京清美联创干细胞科技有限公司 | Kit and method capable of quickly separating adipose tissue-derived stem cells |
| CN105130796A (en) * | 2015-10-22 | 2015-12-09 | 云南民族大学 | Diterpenoid compound and preparing method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| NA LIU，等: "Highly oxygenated ent-pimarane-type diterpenoids from the Chinese Liverwort Pedinophyllum interruptum and their allelopathic activities", 《JOURNAL OF NATURAL PRODUCTS》 * |
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Address after: 810016 Qinghai University, 251 Ning Ning Road, Chengbei District, Qinghai, Xining Applicant after: Zhao Huihui Address before: 211100 Nanjing Medical University, 818 East Tianyuan Road, Jiangning District, Jiangsu, Nanjing Applicant before: Zhao Huihui |
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