CN104450610A - Subculture method of human amniotic mesenchymal stem cells - Google Patents
Subculture method of human amniotic mesenchymal stem cells Download PDFInfo
- Publication number
- CN104450610A CN104450610A CN201410715000.XA CN201410715000A CN104450610A CN 104450610 A CN104450610 A CN 104450610A CN 201410715000 A CN201410715000 A CN 201410715000A CN 104450610 A CN104450610 A CN 104450610A
- Authority
- CN
- China
- Prior art keywords
- cell
- mesenchymal stem
- culture dish
- stem cell
- culture
- 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
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a subculture method of human amniotic mesenchymal stem cells. The subculture method of the human amniotic mesenchymal stem cells comprises the following steps: (1) sucking off a culture liquid in a culture dish after the fusion degree of the human amniotic mesenchymal stem cell subjected to primary culture separation reaches 80-90%, adding PBS for washing, and then sucking off the PBS; (2) adding tryp1E into the culture dish, transferring to a CO2 culture case, hatching for 1-2 minutes, adding a complete medium, and repeatedly blowing and beating until 80-90% of cells are not clung to the wall; (3) centrifuging; (4) discarding a supernatant liquid, re-suspending cell sediment by using a complete medium, blowing, beating and uniformly mixing to obtain a cell suspension, and counting by using a cell counter; and (5) inoculating the cell suspension to the culture dish, adjusting the cell density to be 1.3*10 <4>/cm<2>, then adding the complete medium containing 10ng/ml EGF, transferring the medium to the CO2 culture case to culture. By virtue of the subculture method of human amniotic mesenchymal stem cells, a large quantity of high-activity stem cells can be obtained.
Description
Technical field
Stem cells technology field of the present invention, is specifically related to a kind of human amnion mesenchymal stem cell Secondary Culture method.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs), it is a kind of adult stem cell with the of self-replication capacity and multi-lineage potential, in suitable body or under vitro, can the multiple different cell of differentiation-inducing one-tenth, as adipocyte, scleroblast, chondrocyte, stroma cell, liver cell, myocyte, neurocyte etc.First mescenchymal stem cell finds in marrow, subsequently respectively fat, synovial membrane, bone, muscle, lung, liver, pancreas and in umbilical cord, Cord blood, placenta tissue be separated and prepare mescenchymal stem cell.The injuries of tissues and organs reparation that mescenchymal stem cell can not only cause for old and feeble and pathology as desirable seed cell, goes back hematopoiesis support and promotes that hemopoietic stem cell is implanted, immunity moderation, treat various diseases.
With the most use is at present the mescenchymal stem cell of derived from bone marrow, its weak point is the restriction being subject to age ageing, cell proliferation, differentiation capability reduce, and secondly mesenchymal stem cells MSCs causes immune response when heteroplastic transplantation, affects the research and apply of mescenchymal stem cell.Current research shows, and can be separated from placenta tissue, cultivate and obtain mescenchymal stem cell, be tested and appraised its biological characteristic, known its has very strong proliferation and differentiation ability, and immunogenicity is low.
Existing cultivation amnion mesenchymal stem cell adopts enzyme method, and concrete grammar is: when observation culturing cell adherent growth to 80 ~ 90% converges, inhale and abandon the original substratum of culture dish, draws PBS damping fluid and adds culture dish washing, discard washing lotion; Add (0.5 ~ 0.125%) pancreatin-0.01%EDTA Digestive system, leaching is unrestrained to be covered at the bottom of bottle, observes under inverted microscope; Add FBS (or containing the DMEM/F12 perfect medium of FBS) and stop trysinization; Repeatedly blow and beat flushing, at room temperature centrifugal 5 ~ 10 minutes of about 1000rpm; Abandoning supernatant, adds DMEM/F12 re-suspended cell, by 1: (3 ~ 5) inoculative proportion Secondary Culture; Put 37 DEG C, 5%CO2, cultivate in the cell culture incubator of 95% humidity; When Growth of Cells converges to 80-90%, repeat aforesaid operations and carry out how increasing for cell cultures.
The separation of above-mentioned placenta amnion mescenchymal stem cell with go down to posterity in large scale culturing; in passage process, use pancreatin to carry out cell dissociation; 0.5% ~ 0.125% trypsin digestion cell is used in succeeding generations; usually accurately pancreas enzyme concentration and action time (1min-5min) can not be selected according to cells growth activity state; often to exceed to a certain degree and can not be adherent or dead after causing passage; excessive to cell injury, cell proliferation capacity declines.When cell continuous passage repeatedly, then there will be the cell senescence phenomena of mortality.And inoculum density is according to 1:(3 ~ 5) carry out inoculating cell, in large scale culturing and in going down to posterity, this can increase workload, cannot once collect cell homogeneous in a large number.In addition, within the application algebraically of amnion stem cell was limited in for 6 generations, the application of amnion stem cell is limited greatly.
Therefore be badly in need of setting up a set of effective placenta amnion mescenchymal stem cell large scale culturing technology, meet mescenchymal stem cell at scientific research and the needs in application.This technology belongs to method class technology.
Summary of the invention
Of the present invention being at present sets up a set of effective placenta amnion mescenchymal stem cell large scale culturing technology, provide a kind of human amnion mesenchymal stem cell Secondary Culture method, the method adopts non-enzymic digestion (TrypLE) technology, overcome the damage of enzymolysis to cell, find the optimal inoculum density that goes down to posterity simultaneously, the substratum that design is applicable to, and then the passage number of amnion mesenchymal stem cell is increased to 15 generations more than and still can keeps original dryness, thus meet mescenchymal stem cell in scientific research and the demand in application.
Technical scheme of the present invention is as follows:
A kind of human amnion mesenchymal stem cell Secondary Culture method, is characterized in that comprising the following steps:
(1) raw material cleaning: after the human amnion mesenchymal stem cell degrees of fusion after original cuiture is separated reaches 80 ~ 90%, suck the nutrient solution in culture dish, adds PBS cleaning, then sucks the PBS in culture dish;
(2) digest: in culture dish, add tryplE, be transferred to CO
2after incubator hatches 1-2min, adding perfect medium, repeatedly blow and beat with pasteur pipet, to treating that the cell of 80-90% is no longer adherent, stopping digestion;
(3) centrifugal: step (2) is obtained in cell suspension centrifuge tube, the centrifugal 5min of 1000rpm/min;
(4) count: after centrifugal end, abandoning supernatant, with perfect medium re-suspended cell precipitation, piping and druming mixing, the cell suspension cell counter obtained counts;
(5) bed board and cultivation: by the cell suspension inoculation culture dish of step (4), adjustment cell density is 1.3 × 10
4/ cm
2, then add the perfect medium containing 10ng/ml EGF, substratum gone to CO
2cultivate in cell culture incubator.
Preferably, step (1) PBS liquid adds along culture dish wall, directly do not rinse bottom culture dish.
Preferably, to all around rock after step (1) PBS liquid adds culture dish.
Preferably, in step (2), the minimum amount of tryplE is as the criterion just to cover bottom culture dish.
Preferably, step (2) diameter is the tryplE of 2 ~ 3ml that the culture dish of 10cm adds.
Preferably, described perfect medium is the DMEM/F12 substratum containing 10% bovine serum.
Preferably, step (2) diameter is that the culture dish of 10cm adds 3 ~ 5ml perfect medium.
Preferably, after the cell suspension of step (4) is first dyed blended according to the ratio of 1:1 with trypan blue, inside counting in a hour.
Preferably, after step (5) adds the perfect medium containing EGF, culture dish mixing to be rocked all around.
Preferably, step (5) use is directly the culture dish of 15cm, and the consumption of substratum is 10 ~ 15ml.Use the large culture dish of specification can amplifying cells on a large scale, meet and apply needs.
Compared with prior art, the invention has the beneficial effects as follows:
1, the present invention is in amnion mesenchymal stem cell Secondary Culture process, during passage is cultivated, invent non-enzymatic digestion techniques (TrypLE digestion) and collect adherent fat mesenchymal stem cell, use minimum TrypLE consumption, at utmost reduce the damage to cell in cell dissociation process, search out 1.3 × 10
4/ cm
2best inoculum density culturing cell, use the DMEM/F12 substratum containing 10%, and add the EGF (10ng/ml) of suitable concn in the medium, cultivate on a large scale in the culture dish of φ 15cm, a large amount of stem cells can be obtained, and the vigor of stem cell is high, complete dryness after 15 cultures, is still kept not occur aging phenomenon.
2, the present invention adopts non-enzyme process to collect adherent amnion mesenchymal stem cell, and consumption is low, substantially reduce the damage of cell, the cellular form obtained is good, present fusiformis, bunch group's property growth tendency, and identify through cell counter, cell viability reaches more than 90%, meets passage and condition of storage.
3, the present invention explores the condition of the most applicable large scale culturing, selects 1.3 × 10
4/ cm
2best inoculum density culturing cell, and use the DMEM/F12 substratum containing 10%, one EGF (10ng/ml) adding suitable concn in the medium, cultivate on a large scale in the culture dish of φ 15cm, cultured cells propagation is fast, harvested cell quantity is large, within 2.5 ~ 3.5 days, can grow to 80 ~ 90%.The total cellular score of φ 10cm Tissue Culture Dish results can reach 3 ~ 4 × 10
6, φ 15cm Tissue Culture Dish harvested cell can reach 6 ~ 8 × 10
6.Amnion stem cell can be gone down to posterity for a long time, still keeps complete dryness, do not occur evidence for senescence after 15 generations.
Accompanying drawing explanation
Fig. 1 is the cellular form comparison diagram of non-enzyme process of the present invention and existing enzyme process Secondary Culture;
Wherein, A is existing enzyme process 1st generation MSCs cell (× 100); B is existing enzyme process the 6th generation MSCs cell (× 100); C is non-enzyme process 1st generation MSCs cell (× 100); D is non-enzyme process the 6th generation MSCs cell (× 100), and E is non-enzyme process the 15th generation cell (× 100).
Fig. 2 is the cell proliferation curve comparison figure of non-enzyme process of the present invention and existing enzyme process Secondary Culture.
Fig. 3 is the surface marker flow cytometer detection result of the 1st generation cell that non-enzyme process of the present invention obtains.
Fig. 4 be non-enzyme process of the present invention obtain the 6th generation cell surface marker flow cytometer detection result.
Fig. 5 be non-enzyme process of the present invention obtain the 15th generation cell surface marker flow cytometer detection result.
Fig. 6 is that the present invention's non-enzyme process Secondary Culture P15 becomes fat differentiation state figure for cell.
Fig. 7 is cellular form change (× 100) figure after the amnion mesenchymal stem cell osteogenic induction of the non-enzyme process Secondary Culture of the present invention.
Embodiment
Technical scheme of the present invention is described in further detail, so that the technique effect known the claimed technical scheme of the present invention and can reach below by specific embodiment.
Before introducing specific embodiment, first the term that embodiment relates to is made an explanation:
Pancreatin: 0.25% trypsinase+0.02%EDTA (being mass/volume percentage ratio)
PBS: phosphoric acid buffer
EGF: epidermal growth factor
IBMX:3-isobutyl--1-methylated xanthine
Embodiment 1
A kind of human amnion mesenchymal stem cell Secondary Culture method, concrete grammar is as follows:
(1) selection is observed: take out human amnion mesenchymal stem cell separation and Culture ware, the specification of culture dish is φ 10cm, observes, take pictures under being placed in inverted phase contrast microscope.When cell confluency degree is to 80-90%, passage operation can be carried out.
(2) clean: suck nutrient solution in culture dish with pasteur pipet; Add after appropriate PBS carries out cleaning 2 times in each culture dish with transfer pipet, suck the PBS in culture dish with transfer pipet.Described pasteur pipet range is 3ml, and transfer pipet range is 10ml.The consumption of each cleaning PBS is that 10ml, PBS add along culture dish wall, must not directly rinse bottom culture dish, and the culture dish adding PBS cleaning wants all around to rock, about 10 times.
(3) digest: add tryplE toward culture dish, the tryplE adding 2 ~ 3ml covers bottom culture dish, after transfer culture dish to CO2 incubator hatches 1-2min, observation of cell digestion situation under inverted microscope, add perfect medium subsequently, blow and beat mixing gently, stop digestion.Described substratum is human amnion mesenchymal stem cell substratum (DMEM/F12 mixes according to 1:1,10% foetal calf serum), and the add-on of substratum is 3ml ~ 5ml.Treat that 80-90% cell is no longer adherent, termination digestion can be carried out.
(4) centrifugal: step 3 to be obtained cell suspension 10ml transfer pipet and be transferred in 15ml centrifuge tube, the centrifugal 5min of 1000rpm/min.
(5) count: after centrifugal end, abandoning supernatant, cell precipitation first uses 0.4% trypan blue dyed blended according to 1:1, then precipitates with 5ml perfect medium re-suspended cell.In 1h, piping and druming mixing, gets 20 μ l cell suspension cell counters and counts, and measures total cellular score and cell viability.Described cell counter is adopted as Countstar automated cell calculating instrument.
(6) bed board and cultivation: by cell suspension inoculation in Tissue Culture Dish, adjustment cell density is 1.3 × 104/cm
2, add 10ml perfect medium (φ 10cm culture dish) or 15ml perfect medium (φ 15cm culture dish), add EGF to final concentration 10ng/ml, culture dish mixing 5-10 time is rocked in all around.Be transferred to 5%CO
2, 37 DEG C, saturated humidity is cultivate in the cell culture incubator of 95%.
(7) observe: every 24h observation of cell form and growth degree of converging under an optical microscope after going down to posterity, according to cell growth state, carry out next step passage or frozen and other purposes.
Embodiment 2
After amnion mesenchymal stem cell is separated, obtains P0 for cell, go down to posterity without enzyme process according to the present invention, with the method for traditional enzymolysis, digestion cell in contrast.Continuous passage, observe under an optical microscope 1st generation, the 6th generation, the 15th generation Growth of Cells form.Microscopic observation the results are shown in accompanying drawing 2.
Result shows: in culturing process, and find that by without enzyme process, cellular form is relatively homogeneous, rate of propagation is fast, and adherent speed is fast, is passaged to 15 generations more than, and its form and growth characteristic are also without obvious change.And traditional enzymolysis, digestion passage method, after the 6th generation, cellular form changes, and multiplication rate reduces.And cannot be passaged to for 15 generations.
Embodiment 3
Comparing of the cell proliferation rate of non-enzyme process of the present invention and existing enzyme process Secondary Culture.
Get respectively by non-enzyme process go down to posterity the 3rd generation amnion mesenchymal stem cell, and with existing enzyme process obtain the 3rd generation amnion mesenchymal stem cell, according to 2 × 10
4cells/well is inoculated in 24 orifice plates, digests collection 3 porocyte respectively, by the Trypan Blue living cell counting quantity of 0.4%, calculate mean value, both draftings cell growth curve, the results are shown in accompanying drawing 2 every 48h.
Result shows: the cell doubling time that non-enzyme process obtains is obviously be better than the cell that enzyme process goes down to posterity.
Embodiment 4
The cell surface marker quality testing of non-enzyme process Secondary Culture of the present invention surveys (P1, P2, P3)
(1) get respectively the 1st, 6,15 generation cell, with the cell surface marker between the different algebraically of flow cytomery.Digest collecting cell respectively, get 4 × 106 cells for each batch after counting, packing 4 is managed; Dye solution washes 1 time, the centrifugal 5min of 200g; Abandon supernatant, with dye solution piping and druming mixing cell; Add each 10 μ l of CD45, CD59, CD90 and HLA-DRA antibody, and set a pipe as blank; At 4 DEG C, lucifuge reaction 15-20min; Dye solution is washed once, the centrifugal 5min of 1500rpm; The cell of direct mark abandons supernatant, and lucifuge adds the sample-loading buffer of 500ul, mixing, with 200 eye mesh screen filtration cell samples, and flow cytomery cell-surface antigens.
The surface marker of flow cytomery cell, observe the 1st, 6, the surface marker of the cell in 15 generations obviously do not change.Hematopoietic cell surface marker CD45 (white corpuscle is positive), HLA-DR (MHC-II quasi-molecule) are for all to present feminine gender, and CD59, CD90 all present the positive simultaneously.The amnion mesenchymal stem cell that the third generation is later, cellular form is homogeneous, and purity is more than 90%.
Embodiment 5
The present invention's non-enzyme process Secondary Culture P15 becomes fat Analytical Chemical Experiment for cell
When cell cultures is to the 14th generation, according to 5 × 10
3cell/cm
2be inoculated in six orifice plates, add perfect medium, place 37 DEG C, 5%CO
2incubator is cultivated.Treat that Growth of Cells degree of converging is to about 80-90%, add adipogenic induction substratum and cultivate.Adipogenic induction substratum comprises basic medium DMEM, 10% foetal calf serum, 0.5mM IBMX, 1uM dexamethasone, 100uM indomethacin, 5ug/ml Regular Insulin, 2mm/L glutamine etc., within every three days, changes liquid.Carry out oil red O stain after three weeks, qualification fat drips formational situation.The results are shown in Figure 6.
Result shows: after cultivating three weeks, Microscopic observation amnion mesenchymal stem cell is after oil red O stain, and the cell of about 90% is rich in fat granule, illustrates that mescenchymal stem cell still can have the ability being induced to differentiate into adipocyte.
Embodiment 6
The present invention's non-enzyme process Secondary Culture P15 tests for cell skeletonization
Cell cultures to the 14th generation is when going down to posterity, according to 5 × 10
3cell/cm
2be inoculated in six orifice plates of gelatin coating, add perfect medium, place 37 DEG C, 5%CO
2incubator is cultivated.Treat that Growth of Cells degree of converging is to about 90%, adds Osteogenic Induction Medium and cultivates.Osteogenic Induction Medium comprises: basic medium DMEM/F12,10% foetal calf serum, 50 μMs of xitix, 10mM β-phospho-glycerol, 0.1uM dexamethasone.Every 2-3 days changes liquid, and cell is fixed and Alizarin red staining after 2-3 week.The results are shown in accompanying drawing 7.
Result show: osteogenic induction cultivate the 15th generation mescenchymal stem cell form become cube by fusiformis, there is calcium scoring (Fig. 7-C) after 3 weeks.Dye obvious, cell transforms to scleroblast.Illustrate that stem cell can be induced to differentiate into scleroblast in vitro.
Conclusion: can be learnt by above embodiment, in the Secondary Culture technology of human amnion mesenchymal stem cell, uses traditional Trypsin pancreatin to there is following shortcoming:
1, the large-Trypsin inhibitor of cell injury needs enzyme quick inactivating, and this can cause loss cell or death.
2, freezen protective-Trypsin must be kept at-20 DEG C, through repeatedly multigelation or after preserving for a long time at 4 DEG C, and loss of stability.
3, the large-Trypsin of differences between batches is animal derived, and between batch, otherness is large.
By contrast, TrypLE in the Secondary Culture technology of human amnion mesenchymal stem cell in following advantage:
1, the little-TrypLE of loss cell is the digestive ferment of non-animal source, purer, less to loss cell.Because TrypLE function cells is gentle, do not need Trypsin enzyme inhibitors.
High-the TrypLE of room temperature preservation stability can be stable be stored in room temperature, save reefer space, immediately available facility be provided simultaneously.
Applied range-TrypLE is at the ideal chose having the multiple different clone of cultivating in serum or serum-free environment, can directly substitute 0.25%trypsin edta reagent used in existing step.
The announcement of book and instruction according to the above description, those skilled in the art in the invention can also change above-mentioned embodiment and revise.Therefore, the present invention is not limited to embodiment disclosed and described above, also should fall in the protection domain of claim of the present invention modifications and changes more of the present invention.In addition, although employ some specific terms in this specification sheets, these terms just for convenience of description, do not form any restriction to the present invention.
Claims (10)
1. a human amnion mesenchymal stem cell Secondary Culture method, is characterized in that comprising the following steps:
(1) raw material cleaning: after the human amnion mesenchymal stem cell degrees of fusion after original cuiture is separated reaches 80 ~ 90%, suck the nutrient solution in culture dish, adds PBS cleaning, then sucks the PBS in culture dish;
(2) digest: in culture dish, add tryplE, be transferred to CO
2after incubator hatches 1-2min, add perfect medium, repeatedly blow and beat with pasteur pipet, the cell to 80-90% is no longer adherent, stops digestion;
(3) centrifugal: step (2) is obtained in cell suspension centrifuge tube, the centrifugal 5min of 1000rpm/min;
(4) count: after centrifugal end, abandoning supernatant, with perfect medium re-suspended cell precipitation, piping and druming mixing, the cell suspension cell counter obtained counts;
(5) bed board and cultivation: by the cell suspension inoculation culture dish of step (4), adjustment cell density is 1.3 × 10
4/ cm
2, then add the perfect medium containing 10ng/ml EGF, substratum gone to CO
2cultivate in cell culture incubator.
2. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, is characterized in that: step (1) PBS liquid adds along culture dish wall, does not directly rinse bottom culture dish.
3. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, is characterized in that: will all around rock after step (1) PBS liquid adds culture dish.
4. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, is characterized in that: in step (2), the minimum amount of tryplE is as the criterion just to cover bottom culture dish.
5. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, is characterized in that: step (2) diameter is the tryplE of 2 ~ 3ml that the culture dish of 10cm adds.
6. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, is characterized in that: described perfect medium is the DMEM/F12 substratum containing 10% bovine serum.
7. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, is characterized in that: step (2) diameter is that the culture dish of 10cm adds 3 ~ 5ml perfect medium.
8. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, is characterized in that: after the cell suspension of step (4) is first dyed blended according to the ratio of 1:1 with trypan blue, inside counting in a hour.
9. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, is characterized in that: after step (5) adds the perfect medium containing EGF, will rock culture dish mixing all around.
10. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, is characterized in that: it is directly the culture dish of 15cm that step (5) uses, and the consumption of substratum is 10 ~ 15ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410715000.XA CN104450610B (en) | 2014-11-28 | 2014-11-28 | Subculture method of human amniotic mesenchymal stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410715000.XA CN104450610B (en) | 2014-11-28 | 2014-11-28 | Subculture method of human amniotic mesenchymal stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104450610A true CN104450610A (en) | 2015-03-25 |
| CN104450610B CN104450610B (en) | 2017-05-24 |
Family
ID=52897369
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410715000.XA Active CN104450610B (en) | 2014-11-28 | 2014-11-28 | Subculture method of human amniotic mesenchymal stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104450610B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488628A (en) * | 2017-09-18 | 2017-12-19 | 佛山科学技术学院 | A kind of simple and efficient amniotic fluid stem cell cultural method |
| CN107653223A (en) * | 2017-11-06 | 2018-02-02 | 江西瑞济生物工程技术股份有限公司 | A kind of amnion stem cell media and its cultural method |
| CN110499282A (en) * | 2019-09-03 | 2019-11-26 | 广州赛莱拉干细胞科技股份有限公司 | A kind of culture medium and its application and induction method of the tendon stem cell to Adipocyte Differentiation |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624649A (en) * | 2018-04-09 | 2018-10-09 | 佛山科学技术学院 | A kind of culture medium of the secretion containing amniotic fluid stem cell and its application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130115695A1 (en) * | 2008-11-04 | 2013-05-09 | Viacyte, Inc. | Scalable primate pluripotent stem cell aggregate suspension culture and differentiation thereof |
| CN103451150A (en) * | 2013-05-24 | 2013-12-18 | 北京汉氏联合生物技术有限公司 | Preparation method of placenta source matrix mesenchymal stem cell |
| CN103789258A (en) * | 2012-11-30 | 2014-05-14 | 陆华 | Separation method for human amniotic mesenchymal stem cells |
-
2014
- 2014-11-28 CN CN201410715000.XA patent/CN104450610B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130115695A1 (en) * | 2008-11-04 | 2013-05-09 | Viacyte, Inc. | Scalable primate pluripotent stem cell aggregate suspension culture and differentiation thereof |
| CN103789258A (en) * | 2012-11-30 | 2014-05-14 | 陆华 | Separation method for human amniotic mesenchymal stem cells |
| CN103451150A (en) * | 2013-05-24 | 2013-12-18 | 北京汉氏联合生物技术有限公司 | Preparation method of placenta source matrix mesenchymal stem cell |
Non-Patent Citations (5)
| Title |
|---|
| ABHILOK GARG ET AL.: "Non-enzymatic dissociation of human mesenchymal stromal cells improves chemokine-dependent migration and maintains immunosuppressive function", 《CYTOTHERAPY》 * |
| CATHARINA ELLERSTROM ET AL.: "Derivation of a Xeno-Free Human Embryonic Stem Cell Line", 《STEM CELLS》 * |
| 刘小勇 等: "人羊膜上皮细胞的原代及传代培养", 《广东医学》 * |
| 朱雪敏，等: "绵羊羊膜上皮细胞分离培养及鉴定", 《中国组织工程研究》 * |
| 王秋蛟 等: "人羊膜间充质干细胞的研究进展", 《口腔生物医学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488628A (en) * | 2017-09-18 | 2017-12-19 | 佛山科学技术学院 | A kind of simple and efficient amniotic fluid stem cell cultural method |
| CN107653223A (en) * | 2017-11-06 | 2018-02-02 | 江西瑞济生物工程技术股份有限公司 | A kind of amnion stem cell media and its cultural method |
| CN110499282A (en) * | 2019-09-03 | 2019-11-26 | 广州赛莱拉干细胞科技股份有限公司 | A kind of culture medium and its application and induction method of the tendon stem cell to Adipocyte Differentiation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104450610B (en) | 2017-05-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103805562B (en) | Cultivate the serum free medium of placenta mesenchyma stem cell | |
| CN103154241B (en) | Culture medium composition for culturing amnion-derived mesenchymal stem cell, and method for culturing amnion-derived mesenchymal stem cell by using same | |
| CN101629165B (en) | Preparation method of original mesenchymal stem cell | |
| CN105420183A (en) | Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord Wharton jelly tissue | |
| CN104611292A (en) | Adipose derived stem cell large-scale culture method | |
| CN105713871A (en) | Human chorion mesenchymal stem cell isolated culture method | |
| CN105950550A (en) | Mesenchymal stem cell serum-free medium and cell isolation and cultivation methods | |
| CN104560870A (en) | Method for preparing decidua mesenchymal stem cell | |
| CN105462919A (en) | Method for separating and extracting hUC-MSC (human Umbilical Cord mesenchymal stem cells) from wharton jelly tissue of umbilical cord | |
| CN105420184A (en) | Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord outer layer amnion tissue | |
| CN105238751A (en) | Umbilical cord tissue mesenchymal stem cell isolated culture method | |
| CN103275922B (en) | A kind of fine-wool sheep hair follicle stem cells isolated culture method | |
| CN105420179A (en) | Method for simultaneously extracting epithelial cells and mesenchymal stem cells from umbilical cord and placenta amnion tissues | |
| CN104450611A (en) | Primary separation and culture method of human amniotic mesenchymal stem cells | |
| CN104630142B (en) | A kind of isolation and culture method of ox umbilical cord mesenchymal stem cells | |
| CN108300690A (en) | A kind of isolated culture method and serum free medium of fat mesenchymal stem cell | |
| CN103966159B (en) | Human plactnta Subaerial blue green algae and stem cell bank construction process thereof | |
| CN104560869A (en) | Method for preparing chorionic mesenchymal stem cells | |
| CN104450610B (en) | Subculture method of human amniotic mesenchymal stem cells | |
| CN104651305A (en) | Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells | |
| CN106318906A (en) | Method for large-scale culture of human umbilical cord mesenchymal stem cells | |
| CN103087977A (en) | Culture solution for in vitro efficient amplification of animal cells and application of culture solution | |
| CN104762257A (en) | Method for preparing mesenchymal stem cell from umbilical cord | |
| CN103497892B (en) | A kind of cell cultures base material and its preparation method and application | |
| CN105316279A (en) | Method for efficiently separating and purifying mammary epithelial cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |