CN104450610B - Subculture method of human amniotic mesenchymal stem cells - Google Patents
Subculture method of human amniotic mesenchymal stem cells Download PDFInfo
- Publication number
- CN104450610B CN104450610B CN201410715000.XA CN201410715000A CN104450610B CN 104450610 B CN104450610 B CN 104450610B CN 201410715000 A CN201410715000 A CN 201410715000A CN 104450610 B CN104450610 B CN 104450610B
- Authority
- CN
- China
- Prior art keywords
- culture
- cell
- mesenchymal stem
- culture dish
- stem cell
- 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.)
- Active
Links
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
A kind of stem cells technology field of the present invention, and in particular to human amnion mesenchymal stem cell Secondary Culture method.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs), is a kind of with the of self-replication capacity and many
To the adult stem cell of differentiation potential, under suitable inner or in vitro environment, various different cells can be induced differentiation into,
Such as fat cell, Gegenbaur's cell, cartilage cell, stroma cell, liver cell, myocyte, nerve cell.Mescenchymal stem cell is first
First found in marrow, then respectively in fat, synovial membrane, bone, muscle, lung, liver, pancreas and in umbilical cord, Cord blood, placenta
Mescenchymal stem cell is separated and prepared in tissue.Mescenchymal stem cell not only serves as preferable seed cell for aging and disease
The injuries of tissues and organs reparation that change causes, goes back hematopoiesis support and promotes candidate stem cell implantation, regulation immune, treats various diseases
Disease.
With the most use at present is the mescenchymal stem cell of derived from bone marrow, and it is disadvantageous in that and limited by age ageing
System, cell propagation, differentiation capability reduction, next mesenchymal stem cells MSCs causes immune response in heteroplastic transplantation, between influence
The research of mesenchymal stem cells and application.Current research shows, can be separated from placenta tissue, cultivate that to obtain mesenchyma dry thin
Born of the same parents, by identifying its biological characteristic, it is known that it has very strong Proliferation, Differentiation ability, immunogenicity is low.
Existing culture amnion mesenchymal stem cell uses the enzyme method, specific method to be：Observation cultured cells adherent growth is extremely
80~90% when converging, and the original culture medium of culture dish is abandoned in suction, draws PBS and adds culture dish washing, discards washing lotion；Add
(0.5~0.125%) pancreatin -0.01%EDTA digestive juices, the unrestrained covering bottom of bottle of leaching, observe under inverted microscope；Addition FBS (or
DMEM/F12 complete mediums containing FBS) terminate pancreatin digestion；Piping and druming repeatedly is rinsed, at room temperature 1000rpm or so centrifugations 5
~10 minutes；Abandoning supernatant, adds DMEM/F12 re-suspended cells, by 1: (3~5) inoculative proportion Secondary Culture；37 DEG C are put,
5%CO2, cultivates in the cell culture incubator of 95% humidity；When cell growth is converged to 80-90%, repeating aforesaid operations is carried out
It is many to be expanded for cell culture.
In the separation and passage large-scale culture of above-mentioned placenta amnion mescenchymal stem cell, used during passage
Pancreatin carries out cell dissociation, and 0.5%~0.125% trypsin digestion cell is used in succeeding generations, generally can not accurately according to thin
Intracellular growth vigor state selects pancreas enzyme concentration and action time (1min-5min), and can often exceed causes cell to a certain degree
Can not adherent or death, cell proliferation capacity decline excessive to cellular damage after passage.When cell continuous passage is multiple, then can
There are the cell senescence phenomena of mortality.And inoculum density is according to 1：(3~5) carry out inoculating cell, in large-scale culture and passage, this
Workload can be increased, it is impossible to once collect a large amount of homogeneous cells.In addition, the application algebraically of amnion stem cell be limited in 6 generations with
It is interior, greatly limit the application of amnion stem cell.
Therefore it is badly in need of setting up a set of effective placenta amnion mescenchymal stem cell large-scale culture technology, meets mesenchyma and do
Cell is the need for scientific research is upper with application.The technology belongs to method class technology.
The content of the invention
Of the invention is at present to set up a set of effective placenta amnion mescenchymal stem cell large-scale culture technology, there is provided
A kind of human amnion mesenchymal stem cell Secondary Culture method, the method uses non-enzymic digestion (TrypLE) technology, overcomes enzyme
Damage of the solution to cell, while find the passage inoculum density that is best suitable for, the suitable culture medium of design, and then by amnion mesenchymal
The passage number of stem cell is improved to 15 and more than generation still can keep original dryness, so as to meet mescenchymal stem cell in science
Study and apply upper demand.
Technical scheme is as follows：
A kind of human amnion mesenchymal stem cell Secondary Culture method, it is characterised in that comprise the following steps：
(1) raw material cleaning：After the human amnion mesenchymal stem cell degrees of fusion after original cuiture separation reaches 80~90%,
The nutrient solution in culture dish is sucked, PBS is added, the PBS in culture dish is then sucked；
(2) digest：TryplE is added toward culture dish, CO is transferred to2After incubator is incubated 1-2min, addition is cultivated completely
Base, is blown and beaten repeatedly with pasteur pipet, no longer adherent to the cell for treating 80-90%, terminates digestion；
(3) it is centrifuged：During step (2) obtained into cell suspension centrifuge tube, 1000rpm/min centrifugations 5min；
(4) count：After centrifugation terminates, abandoning supernatant is precipitated with complete medium re-suspended cell, and piping and druming is mixed, and is obtained
Cell suspension counted with cell counter；
(5) bed board and culture：By in the cell suspension inoculation culture dish of step (4), adjustment cell density is 1.3 × 104/
cm2, the complete medium containing 10ng/ml EGF is subsequently adding, culture medium is gone into CO2Cultivated in cell culture incubator.
Preferably, step (1) PBS liquid is added along culture dish wall, and culture dish bottom is not rinsed directly.
Preferably, step (1) PBS liquid will be rocked all around after adding culture dish.
Preferably, the minimum amount of tryplE is defined by just covering culture dish bottom in step (2).
Preferably, the tryplE of 2~3ml that the culture dish of step (2) a diameter of 10cm is added.
Preferably, the complete medium is the DMEM/F12 culture mediums containing 10% cow's serum.
Preferably, the culture dish of step (2) a diameter of 10cm adds 3~5ml complete mediums.
Preferably, the cell suspension of step (4) is first with trypan blue according to 1:After 1 ratio is dyed blended, in one hour
Count.
Preferably, after step (5) adds the complete medium containing EGF, culture dish mixing is all around rocked.
Preferably, using being directly the culture dish of 15cm, the consumption of culture medium is 10~15ml to step (5).Use specification
Big culture dish can amplifying cells on a large scale, satisfaction applies needs.
Compared with prior art, the beneficial effects of the invention are as follows：
1st, the present invention in passage culture, has invented non-enzymatic and has disappeared during amnion mesenchymal stem cell Secondary Culture
Change technology (TrypLE digestion) collects adherent fat mesenchymal stem cell, using minimum TrypLE consumptions, at utmost subtracts
Damage during few cell dissociation to cell, searches out 1.3 × 104/cm2Optimal inoculum density cultured cells, using containing 10%
DMEM/F12 culture mediums, and in the medium add suitable concn EGF (10ng/ml), enter in the culture dish of φ 15cm
The large-scale culture of row, is obtained in that substantial amounts of stem cell, and the vigor of stem cell is high, 15 are commissioned to train support after remain in that it is complete
Dryness occurs without aging phenomenon.
2nd, the present invention collects adherent amnion mesenchymal stem cell using non-enzyme process, and consumption is low, substantially reduces cell
Damage, the cellular morphology of acquisition is good, and fusiformis, cluster growth tendency is presented, and identified through cell counter, and cell is lived
Power reaches more than 90%, meets passage and condition of storage.
3rd, the present invention explores the condition for being best suitable for large-scale culture, selection 1.3 × 104/cm2Optimal inoculum density culture
Cell, and using containing 10% DMEM/F12 culture mediums, one adds the EGF (10ng/ml) of suitable concn in the medium, in φ
Cultivated on a large scale in the culture dish of 15cm, the cell propagation of culture is fast, and harvesting quantity is big, 2.5~3.5 days
Grow to 80~90%.The TCS that φ 10cm Tissue Culture Dish is harvested is up to 3~4 × 106, φ 15cm Tissue Culture Dish results
Cell is up to 6~8 × 106.Amnion stem cell can be passed on for a long time, and 15 instead of remain in that complete dryness afterwards, do not go out
Existing evidence for senescence.
Brief description of the drawings
Fig. 1 is the cellular morphology comparison diagram of non-enzyme process of the invention and existing enzyme process Secondary Culture；
Wherein, A is existing enzyme process 1st generation MSCs cells (× 100)；B is existing enzyme process the 6th generation MSCs cell (× 100)；
C is non-enzyme process 1st generation MSCs cells (× 100)；D is non-enzyme process the 6th generation MSCs cell (× 100), and E is that the generation of non-enzyme process the 15th is thin
Born of the same parents (× 100).
Fig. 2 is the cell growth curve comparison diagram of non-enzyme process of the 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 invention is obtained.
Fig. 4 is the surface marker flow cytometer detection result of the 6th generation cell that non-enzyme process of the invention is obtained.
Fig. 5 is the surface marker flow cytometer detection result of the 15th generation cell that non-enzyme process of the invention is obtained.
Fig. 6 is non-enzyme process Secondary Culture P15 of the invention for cell into fat differentiation state figure.
Fig. 7 be non-enzyme process Secondary Culture of the invention amnion mesenchymal stem cell osteogenic induction after cellular morphology change (×
100) figure.
Specific embodiment
Technical scheme is described in further detail below by specific implementation example, so as to the clear present invention
Claimed technical scheme and the technique effect that can be reached.
Before specific implementation example is introduced, first the term that embodiment is related to is explained：
Pancreatin：0.25% trypsase+0.02%EDTA (being mass/volume percentage)
PBS:Phosphate buffer
EGF:Epidermal growth factor
IBMX：3- isobutyl group -1- methylated xanthines
Embodiment 1
A kind of human amnion mesenchymal stem cell Secondary Culture method, specific method is as follows：
(1) selection is observed：Take out human amnion mesenchymal stem cell and be separately cultured ware, the specification of culture dish is φ 10cm, is put
Observed under inverted phase contrast microscope, taken pictures.When cell confluency degree to 80-90%, you can carry out passage operation.
(2) clean：Nutrient solution in culture dish is sucked with pasteur pipet；With pipette in each culture dish plus appropriate
After PBS carries out cleaning 2 times, the PBS in culture dish is sucked with pipette.The pasteur pipet range is 3ml, and pipette range is
10ml.The consumption of cleaning PBS is 10ml every time, and PBS is added along culture dish wall, must not directly rinse culture dish bottom, is added
The culture dish of PBS will be rocked around, 10 times or so.
(3) digest：TryplE is added toward culture dish, the tryplE covering culture dishes bottom of 2~3ml, transfer culture is added
After ware is incubated 1-2min to CO2 incubators, observation of cell digestion situation, is subsequently added complete medium under inverted microscope,
Gently piping and druming is mixed, and terminates digestion.The culture medium is that (DMEM/F12 is according to 1 for human amnion mesenchymal stem cell culture medium：1 mixes
With 10% hyclone), the addition of culture medium is 3ml~5ml.Treat that 80-90% cells are no longer adherent, can be terminated
Digestion.
(4) it is centrifuged：Step 3 is obtained into cell suspension 10ml pipettes to be transferred in 15ml centrifuge tubes, 1000rpm/min from
Heart 5min.
(5) count：After centrifugation terminates, abandoning supernatant, cell precipitation is first with 0.4% trypan blue according to 1：1 is dyed blended,
Precipitated with 5ml complete mediums re-suspended cell again.Blown and beaten in 1h and mixed, take the counting of 20 μ l cell suspensions cell counters,
Determine TCS and cell viability.The cell counter is adopted as Countstar automated cell calculating instruments.
(6) bed board and culture：By in cell suspension inoculation to Tissue Culture Dish, adjustment cell density is 1.3 × 104/
cm2, 10ml complete mediums (φ 10cm culture dishes) or 15ml complete mediums (φ 15cm culture dishes) are added, add EGF
To final concentration 10ng/ml, culture dish is rocked around and is mixed 5-10 times.It is transferred to 5%CO2, 37 DEG C, saturated humidity be
Cultivated in 95% cell culture incubator.
(7) observe：After passage every 24h under an optical microscope observation of cell form and growth degree of converging, according to cell
Growth conditions, carry out the passage of next step or freeze and other purposes.
Embodiment 2
After amnion mesenchymal stem cell is separated, P0 is obtained for cell, passed on without enzyme process according to the present invention, with
The method of traditional enzymolysis, digestion cell is used as control.Continuous passage, observes 1st generation, the 6th generation, the 15th generation under an optical microscope
Cell growth form.Microscopic observation result is shown in accompanying drawing 2.
Result shows：In incubation, find by the way that without enzyme process, cellular morphology is relatively uniform, growth rate is fast, adherent speed
Degree is fast, is passaged to 15 more than generation, and its form and growth characteristic are also without substantially change.And traditional enzymolysis, digestion passage method,
Cellular morphology changes after the 6th generation, multiplication rate reduction.And cannot be passaged to for 15 generations.
Embodiment 3
The comparing of non-enzyme process of the invention and the cell proliferation rate of existing enzyme process Secondary Culture.
The 3rd generation amnion mesenchymal stem cell passed on by non-enzyme process is taken respectively, and the 3rd generation sheep obtained with existing enzyme process
Intermembranous mesenchymal stem cells, according to 2 × 104Cells/well is inoculated in 24 orifice plates, collects 3 hole cells every 48h digestion respectively, is used
0.4% Trypan Blue living cell counting quantity, calculates average value, draws both cell growth curves, as a result sees accompanying drawing 2.
Result shows：The cell doubling time that non-enzyme process is obtained is the cell for being substantially better than enzyme process passage.
Embodiment 4
The cell surface marker analyte detection (P1, P2, P3) of non-enzyme process Secondary Culture of the invention
(1) the 1st, 6,15 generation cells are taken respectively, with the cell surface marker between flow cytomery difference algebraically.Point
Cell Xiao Hua not be collected, each batch takes 4 × 106 cells after counting, packing 4 is managed；Dye solution is washed 1 time, 200g centrifugations
5min；Supernatant is abandoned, is blown and beaten with dye solution and is mixed cell；Each 10 μ l of CD45, CD59, CD90 and HLA-DRA antibody are added, and
If a pipe is blank；At 4 DEG C, lucifuge reaction 15-20min；Dye solution is washed once, 1500rpm centrifugations 5min；Directly
The cell for connecing mark abandons supernatant, and lucifuge adds the sample-loading buffer of 500ul, mixes, with 200 eye mesh screen filtration cell samples, stream
Formula cell instrument detects cell surface antigen.
The surface marker of flow cytomery cell, it was observed that the 1st, 6, the surface marker of the cell in 15 generations it is not bright
It is aobvious to change.Hematopoietic cell surface marker CD45 (leucocyte is positive), HLA-DR (MHC-II quasi-molecules) are negative to present, while
CD59, CD90 are presented the positive.The later amnion mesenchymal stem cell of the third generation, cellular morphology is homogeneous, and purity is more than 90%.
Embodiment 5
The non-enzyme process Secondary Culture P15 of the present invention is for cell into fat Analytical Chemical Experiment
During cell culture to 14 generation, according to 5 × 103Cell/cm2It is inoculated in six orifice plates, adds complete medium, puts
Put 37 DEG C, 5%CO2Incubator culture.Treat that cell growth degree of converging to 80-90% or so, adds adipogenic induction culture medium to carry out
Culture.Adipogenic induction culture medium include basal medium DMEM, 10% hyclone, 0.5mM IBMX, 1uM dexamethasone,
100uM Indomethacins, 5ug/ml insulin, 2mm/L glutamine etc., change liquid in every three days.Oil red O stain is carried out after three weeks, is reflected
Determine fat drips formational situation.Result is shown in Fig. 6.
Result shows：After cultivating three weeks, Microscopic observation amnion mesenchymal stem cell by after oil red O stain, about 90%
Cell is rich in fat drop, illustrates that mescenchymal stem cell can still have the ability for being induced to differentiate into fat cell.
Embodiment 6
The non-enzyme process Secondary Culture P15 of the present invention is tested for cell skeletonization
When cell culture to the 14th generation is passed on, according to 5 × 103Cell/cm2It is inoculated in six orifice plates of gelatin coating,
Complete medium is added, 37 DEG C, 5%CO is placed2Incubator culture.Cell growth degree of converging to 90% or so is treated, skeletonization is added
Inducing culture is cultivated.Osteogenic Induction Medium includes：Basal medium DMEM/F12,10% hyclone, 50 μM resist
Bad hematic acid, 10mM β-phosphoglycerol, 0.1uM dexamethasone.Per liquid is changed within 2-3 days, cell was fixed and alizarin red after 2-3 weeks
Dyeing.Result is shown in accompanying drawing 7.
Result shows：15th generation mescenchymal stem cell form of osteogenic induction culture becomes cubic by fusiformis, goes out after 3 weeks
Existing calcium scoring (Fig. 7-C).Dyeing is obvious, and cell is converted to Gegenbaur's cell.Illustrate that stem cell can induce in vitro
It is divided into Gegenbaur's cell.
Conclusion：By above example it is known that in the Secondary Culture technology of human amnion mesenchymal stem cell, using
Traditional Trypsin pancreatin has the following disadvantages：
1st, the big-Trypsin inhibitor of cellular damage is needed enzyme quick inactivating, and this can cause loss cell or death.
2nd, freezen protective-Trypsin must be maintained in -20 DEG C, be preserved by multiple multigelation or at 4 DEG C long-term
Afterwards, loss of stability.
3rd, the big-Trypsin of differences between batches is animal derived, and otherness is big between batch.
By contrast, TrypLE in the Secondary Culture technology of human amnion mesenchymal stem cell in advantages below：
1st, the small-TrypLE of loss cell is the digestive ferment of non-animal source, purer, smaller to loss cell.Due to
TrypLE function cells are gentle, it is not necessary to Trypsin enzyme inhibitors.
What room temperature preservation stability-TrypLE high can stablize is stored in room temperature, saves reefer space, while providing
Instant available facility.
- the TrypLE that has a wide range of application is the ideal of the various different cell lines cultivated in having serum or serum-free environment
Selection, can directly substitute 0.25%trypsin edta reagents used in existing step.
The announcement and teaching of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula is changed and changed.Therefore, the invention is not limited in specific embodiment disclosed and described above, to of the invention
Some modifications and changes should also be as falling into scope of the claims of the invention.Although additionally, being used in this specification
Some specific terms, but these terms are merely for convenience of description, do not constitute any limitation to the present invention.
Claims (8)
1. a kind of human amnion mesenchymal stem cell Secondary Culture method, it is characterised in that comprise the following steps：
(1) raw material cleaning：After the human amnion mesenchymal stem cell degrees of fusion after original cuiture separation reaches 80~90%, suck
Nutrient solution in culture dish, adds PBS, then sucks the PBS in culture dish；
(2) digest：TryplE is added toward culture dish, CO is transferred to2After incubator is incubated 1-2min, complete medium is added, used
Pasteur pipet is blown and beaten repeatedly, and the cell to 80-90% is no longer adherent, terminates digestion；
(3) it is centrifuged：During step (2) obtained into cell suspension centrifuge tube, 1000rpm/min centrifugations 5min；
(4) count：After centrifugation terminates, abandoning supernatant is precipitated with complete medium re-suspended cell, and piping and druming is mixed, and what is obtained is thin
Born of the same parents' suspension is counted with cell counter；
(5) bed board and culture：By in the cell suspension inoculation culture dish of step (4), adjustment cell density is 1.3 × 104/cm2,
The complete medium containing 10ng/ml EGF is subsequently adding, culture medium is gone into CO2Cultivated in cell culture incubator,
Wherein, the consumption of tryplE is the tryplE of 2~3ml of the culture dish addition of 10cm in the step (2).
2. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, it is characterised in that：Step (1) PBS liquid
It is to be added along culture dish wall, culture dish bottom is not rinsed directly.
3. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, it is characterised in that：Step (1) PBS liquid
All around to be rocked after adding culture dish.
4. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, it is characterised in that：It is described to cultivate completely
Base is the DMEM/F12 culture mediums containing 10% cow's serum.
5. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, it is characterised in that：Step (2) diameter
For the culture dish of 10cm adds 3~5ml complete mediums.
6. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, it is characterised in that：Step (4) it is thin
Born of the same parents' suspension is first with trypan blue according to 1:After 1 ratio is dyed blended, in inside counting in a hour.
7. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, it is characterised in that：Step (5) is added
After complete medium containing EGF, culture dish mixing is all around rocked.
8. human amnion mesenchymal stem cell Secondary Culture method as claimed in claim 1, it is characterised in that：Step (5) is used
It is directly the culture dish of 15cm, the consumption of culture medium 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 CN104450610A (en) | 2015-03-25 |
| CN104450610B true 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 (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 |
Families Citing this family (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 |
| CN110499282B (en) * | 2019-09-03 | 2021-05-11 | 广州赛莱拉干细胞科技股份有限公司 | Culture medium and application thereof, and method for inducing tendon stem cells to differentiate into adipocytes |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8895300B2 (en) * | 2008-11-04 | 2014-11-25 | 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 |
| CN103451150B (en) * | 2013-05-24 | 2015-10-14 | 北京汉氏联合生物技术有限公司 | A kind of preparation method of placenta source matrix mesenchymal stem cell |
-
2014
- 2014-11-28 CN CN201410715000.XA patent/CN104450610B/en active Active
Cited By (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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104450610A (en) | 2015-03-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104611292B (en) | A kind of fat mesenchymal stem cell large-scale cultivation method | |
| CN105420183A (en) | Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord Wharton jelly tissue | |
| CN104450610B (en) | Subculture method of human amniotic mesenchymal stem cells | |
| CN104450611A (en) | Primary separation and culture method of human amniotic mesenchymal stem cells | |
| CN105950550A (en) | Mesenchymal stem cell serum-free medium and cell isolation and cultivation methods | |
| CN105670986A (en) | Culture medium for inducing human umbilical cord mesenchymal stem cells to differentiate into islet-like cells and induction method therefor | |
| CN105420184A (en) | Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord outer layer amnion tissue | |
| CN105462919A (en) | Method for separating and extracting hUC-MSC (human Umbilical Cord mesenchymal stem cells) from wharton jelly tissue of umbilical cord | |
| CN104651305A (en) | Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells | |
| CN108300690A (en) | A kind of isolated culture method and serum free medium of fat mesenchymal stem cell | |
| CN106318906A (en) | Method for large-scale culture of human umbilical cord mesenchymal stem cells | |
| CN107385517A (en) | The construction method of mesenchyma stem cell | |
| CN105850979A (en) | Cryoprotective solution and cryopreservation method for bone mesenchymal stem cells | |
| CN106119191A (en) | A kind of placental villi plate mescenchymal stem cell and clinicization preparation method | |
| CN104630142A (en) | Separation and culture method of bovine umbilical cord mesenchymal stem cells | |
| CN105420185A (en) | Method for separating and extracting hUC-MSC from umbilical cord outer layer amnion tissue | |
| CN104726401A (en) | Method for improving success rate of umbilical cord blood mesenchymal stem cell culture by using placental mesenchymal stem cells | |
| CN109628388B (en) | Isolation of mesenchymal stem cells from placental blood vessels with digestive enzyme composition | |
| CN105441386A (en) | Culture and identification method for very small porcine embryonic-like stem cells | |
| CN107236705B (en) | Human placenta chorion mesenchymal stem cell culture system | |
| CN105886462A (en) | Composition ADSCs for ADSCs culture and ADSCs culture method | |
| CN105420187B (en) | A kind of kit for cultivating hUC-MSC step by step and the hUC-MSC using kit acquisition | |
| CN106479970A (en) | A kind of method of large-scale culture human adipose mesenchymal stem cells | |
| CN106591230A (en) | Human umbilical cord mesenchymal stem cell culture solution and culture method thereof | |
| CN106032529A (en) | Method for in-vitro separation, amplification and induced differentiation of placenta-derived mesenchymal stem 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 |