CN105087474A - Culture method of deciduous tooth pulp stem cells - Google Patents
Culture method of deciduous tooth pulp stem cells Download PDFInfo
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- CN105087474A CN105087474A CN201510209690.6A CN201510209690A CN105087474A CN 105087474 A CN105087474 A CN 105087474A CN 201510209690 A CN201510209690 A CN 201510209690A CN 105087474 A CN105087474 A CN 105087474A
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Abstract
The invention relates to the field of stem cells and discloses a culture method of deciduous tooth pulp stem cells. The culture method includes: taking pulp tissue, cutting the pulp tissue into pieces, and then adding I-type collagenase; digesting for 10-20min under conditions of 37 DEG C and 200rpm; stopping digestion of a serum-free culture medium; blowing and beating discrete cell mass to obtain single discrete cells; adding cell suspension to re-suspend the cells, and adjusting cell density; culturing the cells in a carbon dioxide incubator at temperature of 37 DEG C and with humidity of 95%; when the cells grow to be fused by 80-90%, using digestive liquid of trypsase to digest the cells before passage culture, wherein the cell suspension is composed of the serum-free culture medium, epidermal growth factor and basic fibroblast growth factor. The cells cultured by the method are good in shape, have tendency of fusiform cluster growing and are high in activity and quick in proliferation, good stem cell characteristics of the deciduous tooth pulp stem cells can be maintained, and the culture method is suitable for large-scale culture of the deciduous tooth pulp stem cells.
Description
Technical field
The present invention relates to stem cell field, be specifically related to a kind of cultural method of dental pulp stem cell, especially relate to a kind of cultural method of deciduous teeth dental pulp stem cell.
Background technology
Miura in 2003 etc. have found a kind of pluripotent stem cell in the pulp tissue of deciduous teeth that comes off, compare with permanent teeth dental pulp stem cell with mesenchymal stem cells MSCs, it has higher proliferation rate, population doublings ability, clonality and height differentiation capability, by its called after deciduous teeth dental pulp stem cell (stemcellsfromdeciduousteeth, SHED).Human milk tooth dental pulp stem cell is mescenchymal stem cell (mesenchymalstemcells, MSCs) one, there is the mescenchymal stem cell of the of self-replication capacity and multi-lineage potential, differentiation-inducingly can become dentine, scleroblast, chondrocyte, stearoblast, neurocyte, liver cell, myocyte etc.
The mankind have 20 deciduous teeth, can naturally come off gradually in the newborn permanent teeth phase of replacing, and therefore pulp tissue source fairly obtains, and hurtless measure.Pulp tissue comes from autologous tissue, without immunological rejection, has higher security.Biological characteristics in view of the above, dental pulp stem cell has broad application prospects in treatment bone and dental tissue Regeneration and Repair, nervous system disorders and amyotrophy, disease of immune system, skin injury, hepatic diseases etc.
Application number is the scale production process that the Chinese patent of 201210514497.X discloses a kind of dental pulp stem cell.In literary composition just like
following tablestate: in step (1), pulp tissue aseptically selects, and shreds with after wash buffer; The digestion of pulp tissue adopts the 0.1%-0.6% collagenase of 5-10 times of volume, in 37 DEG C of incubators, stop digestion after shaking culture 30-180min with the cell culture fluid containing foetal calf serum, described cell culture fluid is selected from commercial D-MEM, DMEM substratum containing 10-20% foetal calf serum and serum free medium.But the method enzymolysis time is long, tissue digestion is excessive, and it is few that dental pulp stem cell cell obtains quantity.And the method is taked to cultivate containing the DMEM of foetal calf serum, foetal calf serum has source of pollution, poor stability between batch, is unfavorable for that cell is in animal model.
Summary of the invention
In view of this, the object of the invention is for prior art Problems existing, a kind of cultural method of deciduous teeth dental pulp stem cell is provided, cultural method cultured cells form of the present invention is good, present fusiformis, bunch group's property growth tendency, cytoactive is high, simple economy, a large amount of high purity deciduous teeth dental pulp stem cell can be obtained, and the stem cell properties that deciduous teeth dental pulp stem cell is good can be kept.
In order to realize object of the present invention, the present invention adopts following technical scheme:
A kind of cultural method of deciduous teeth dental pulp stem cell, get pulp tissue, add type i collagen enzyme after shredding, 37 DEG C, 200rpm digests 10-20min, serum free medium stops digestion, piping and druming discrete cellular agglomerate obtains single discrete cell, adds cell suspension re-suspended cell and adjusts cell density, in 37 DEG C, humidity is cultivate in the CO2gas incubator of 95%, when Growth of Cells merges to 80%-90%, use containing Secondary Culture after tryptic Digestive system peptic cell; Wherein said cell suspension is made up of serum free medium, Urogastron, Basic Fibroblast Growth Factor.
The cultural method of deciduous teeth dental pulp stem cell of the present invention adopts type i collagen enzyme can obtain more cell quantity at 37 DEG C of enzymolysis, digestion pulp tissue 10-20min.Cultural method whole process of the present invention takes serum-free culture cultivate and go down to posterity simultaneously, avoids the DMEM culture medium culturing adopting foetal calf serum.
In some embodiments, the add-on of type i collagen enzyme described in cultural method of the present invention is 10-20 times of volume of pulp tissue.
In some preferred embodiments, the concentration of type i collagen enzyme described in cultural method of the present invention is 0.3% ~ 0.5%.I.e. 3g/L ~ 5g/L.
The cultural method of deciduous teeth dental pulp stem cell of the present invention, the deciduous incisor tooth not having tooth prosthesis change and necrosis that the children that described deciduous teeth can take from family members' agreement pull out because of delay, be put in cold being equipped with of 4 DEG C of chances after pulling out immediately to preserve containing in dual anti-PBS centrifuge tube, take out in 24 hours and use.Described is the phosphate buffered saline buffer containing penicillin and Streptomycin sulphate containing dual anti-PBS, and wherein penicillin working concentration is 100U/mL, and Streptomycin sulphate working concentration is 0.1mg/mL.
Tooth described in the cultural method of deciduous teeth dental pulp stem cell of the present invention rinses three times repeatedly with containing dual anti-PBS solution in advance, is wrapped in by tooth with clamp schizodont tooth in sterile gauze, Exposed Pulp tissue; With aseptic nipper gripping pulp tissue, the pulp tissue of excision root tip 1mm.
Described in the cultural method of deciduous teeth dental pulp stem cell of the present invention, pulp tissue is according to pulp tissue's size, with ophthalmology curved scissors, pulp tissue is cut into 1mm
3.
In some embodiments, described cultural method also comprises the step of cleaning single discrete cellular.
In some preferred embodiments, described in described cultural method, cleaning is specially centrifugal rear PBS washing and precipitating, centrifugal collecting precipitation.In certain embodiments, described centrifugal be the centrifugal 3-5min of 1000rpm-2000rpm.
In some embodiments, diameter≤70 μm of single discrete cell described in described cultural method.In certain embodiments, the method for the single discrete cellular of described acquisition is specially piping and druming discrete cellular agglomerate, by the cell screen filtration of 70 μm.
In some embodiments, cell density described in described cultural method is 2 × 10
3/ cm
2-5 × 10
3/ cm
2.With 2 × 10
3/ cm
2-5 × 10
3/ cm
2cell density inoculation, cell P1 generation can increase 20 times than primary cell number.
In some embodiments, described in described cultural method, the final concentration of Urogastron described in cell suspension is 10ng/mL.
In some embodiments, described in described cultural method, the final concentration of Basic Fibroblast Growth Factor described in cell suspension is 10ng/mL.
In some embodiments, a cell suspension was changed every three days in culturing process described in described cultural method.
In some embodiments, tryptic concentration described in described cultural method is 0.125%.
In a specific embodiment, with application number disclosed in 201210514497.X method cultured cells as a comparison case, observe the cell that the method for embodiment 1 is cultivated 7 days, cytoactive is high, and cell viability reaches more than 90%; Cell proliferation is fast, and harvested cell quantity is large, and primary separation can grow to 80-90% in 7-8 days; Only need to go down to posterity in 3-5 days, cell number once just can be made to increase more than 20 times.
In a specific embodiment, observation of cell surface marker expression.The cultural method that result shows deciduous teeth dental pulp stem cell of the present invention cultivate deciduous teeth dental pulp stem cell 1st generation, the 6th generation, the 10th generation the surface marker of cell obviously do not change.Deciduous teeth dental pulp stem cell surface marker CD45 (white corpuscle is positive), HLA-DR (MHC-II quasi-molecule) are for all to present feminine gender, and mescenchymal stem cell surface marker CD59, CD90 all presents the positive simultaneously.The deciduous teeth dental pulp stem cell that the third generation is later, cellular form is homogeneous, and purity is more than 98%.
In a specific embodiment, observing P3 becomes fat to break up situation for cell induction.Result shows, and the deciduous teeth dental pulp stem cell cell of the cultural method cultivation of deciduous teeth dental pulp stem cell of the present invention, after induction, carries out oil red O stain, and visible significantly fat drips formation.
In a specific embodiment, P3 is observed for cell induction Osteoblast Differentiation situation.Result shows, and compared with control group, the deciduous teeth dental pulp stem cell cell that the cultural method of deciduous teeth dental pulp stem cell of the present invention is cultivated has obvious calcium tubercle.Show that scleroblast is formed.
The cultural method of deciduous teeth dental pulp stem cell of the present invention, get pulp tissue, add type i collagen enzyme after shredding, 37 DEG C, 200rpm digests 10-20min, serum free medium stops digestion, piping and druming discrete cellular agglomerate obtains single discrete cell, adds cell suspension re-suspended cell and adjusts cell density, in 37 DEG C, humidity is cultivate in the CO2gas incubator of 95%, when Growth of Cells merges to 80%-90%, use containing Secondary Culture after tryptic Digestive system peptic cell; Wherein said cell suspension is made up of serum free medium, Urogastron, Basic Fibroblast Growth Factor.Cultural method of the present invention is simple to operate, safe and effective.Experiment shows, compared with the conventional method, the cultural method culturing cell form of deciduous teeth dental pulp stem cell of the present invention is good, presents fusiformis, bunch group's property growth tendency; Cytoactive is high, and cell is identified through cell counter, and cell viability reaches more than 90%; Cell proliferation is fast, and harvested cell quantity is large, and primary separation can grow to 80-90% in 7-8 days; Only need to go down to posterity in 3-5 days, cell number once just can be made to increase more than 20 times; And the form of deciduous teeth dental pulp stem cell and good stem cell properties can be kept very well, be applicable to the mass propgation of deciduous teeth dental pulp stem cell.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, to use to required in embodiment or description of the prior art below
accompanying drawingbe briefly described.
fig. 1show that embodiment 2 cell viability detects
figure;
fig. 2show embodiment 3 cultured cells state
figure, wherein
figure afor application number method disclosed in 201210514497.X cultivates the cell of 7 days
figure,
figure bfor method described in embodiment 1 cultivates the cell of 7 days
figure, magnification is 40 times;
fig. 3show embodiment 4 cell expansion culturing cell sum statistics
figure;
fig. 4show the expression of results of embodiment 5 1st generation cell surface marker
figure, wherein
figure a-c is 1st generation compared with control cells group,
figure dthe 1st generation experimental cell group that-f cultivates for method described in embodiment 1;
figure afor FSC and SSC expression
figure,
figure bfor HLA-DR and CD59 expression
figure,
figure cfor CD45 and CD90 expression
figure,
figure dfor FSC and SSC expression
figure,
figure efor HLA-DR and CD59 expression
figure,
figure ffor CD45 and CD90 expression
figure;
fig. 5show the expression of results of embodiment 5 the 6th generation cell surface marker
figure, wherein
figure a-c be the 6th generation compared with control cells group,
figure d-f be described in embodiment 1 method cultivate the 6th generation experimental cell group;
figure afor FSC and SSC expression
figure,
figure bfor HLA-DR and CD59 expression
figure,
figure cfor CD45 and CD90 expression
figure,
figure dfor FSC and SSC expression
figure,
figure efor HLA-DR and CD59 expression
figure,
figure ffor CD45 and CD90 expression
figure;
fig. 6show the expression of results of embodiment 5 the 10th generation cell surface marker
figure, wherein
figure a-c be the 10th generation compared with control cells group,
figure d-f be described in embodiment 1 method cultivate the 10th generation experimental cell group;
figure afor FSC and SSC expression
figure,
figure bfor HLA-DR and CD59 expression
figure,
figure cfor CD45 and CD90 expression
figure,
figure dfor FSC and SSC expression
figure,
figure efor HLA-DR and CD59 expression
figure,
figure ffor CD45 and CD90 expression
figure;
fig. 7show that embodiment 6P3 becomes fat differentiated result for cell induction
figure, wherein
figure afor application number method disclosed in 201210514497.X cultivate the cellular control unit oil red O stain of 28 days after fat drip formational situation
figure,
figure bwith
figure cfor method described in embodiment 1 cultivate the experimental group cell oil red O stain of 28 days after fat drip formational situation
figure; Magnification is 40 times;
fig. 8show that embodiment 7P3 is for cell induction Osteoblast Differentiation result
figure, wherein
figure athe cellular control unit VonKossa cultivated 21 days for application number method disclosed in 201210514497.X dyes
figure,
figure bthe experimental group cell VonKossa cultivated 21 days for method described in embodiment 1 dyes
figure; Magnification is 40 times.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The cultivation of embodiment 1, human milk tooth dental pulp stem cell
(1) after family members agree to, collect the deciduous incisor tooth that children clinically pull out because of delay, require do not have tooth prosthesis to become and necrosis.Be put in 4 DEG C after pulling out immediately to meet and cold be equipped with containing in dual anti-PBS centrifuge tube, take out in 24 hours and use.
(2) take out tooth, utilize and repeatedly rinse three times containing dual anti-PBS solution, tooth is wrapped in clamp schizodont tooth in sterile gauze, Exposed Pulp tissue; With aseptic nipper gripping pulp tissue, the pulp tissue of excision root tip 1mm.According to pulp tissue's size, with ophthalmology curved scissors, pulp tissue is cut into 1mm
3, be placed in 50mL centrifuge tube.
(3) add the 5g/LI Collagenase Type of 10 times of volumes, fully mix after sealing, be transferred in Tempeerature-constant air shaking table, 37 DEG C, 200R digests about 10-20min.
(4) add isopyknic serum free medium and stop digestion, repeatedly blow and beat discrete cellular agglomerate, by the cell screen filtration of 70 μm, to obtain single discrete cell, the centrifugal 3-5min of 1000rpm-2000rpm.
(5) supernatant is abandoned, by 30mLPBS washing and precipitating 1 time, the centrifugal 3-5min of 1000rpm-2000rpm.
(6) precipitation serum free medium is resuspended, gets 20 μ L cell suspensions, according to cell suspension (V): 0.4% trypan blue (V)=3:1 ratio mixing, calculates cell viability in Auto-counting of Cells instrument.
(7) with 0.5 × 10
4/ cm
2~ 1 × 10
4/ cm
2be seeded in six orifice plates, every hole adds 2mL serum free medium, add Urogastron EGF and Basic Fibroblast Growth Factor bFGF (both are 10ng/mL by final concentration) again, mixing cell suspension, is put in 37 DEG C, humidity is cultivate in the CO2gas incubator of 95%.
(8) every 3d changes liquid 1 time, and about 7-14d can observe the formation of clone under the microscope, and when Growth of Cells converges to 80%-90%, 0.125% trypsin digestion and cell, carries out Secondary Culture.
Embodiment 2, cell viability detect
With application number, disclosed in 201210514497.X, method cultured cells as a comparison case (control group), according to method (sample sets) isolated cell of embodiment 1, is collected isolated cell and calculate cell viability in Auto-counting of Cells instrument.Result
as Fig. 1.
By
fig. 1the vigor of the dental pulp stem cell that the cultural method of the visible deciduous teeth dental pulp stem cell of the present invention of result obtains can reach more than 90%.
Embodiment 3, cellular form are observed
With application number, disclosed in 201210514497.X, method cultured cells as a comparison case (control group), is observed the cellular form that the method (sample sets) of embodiment 1 cultivates the cell of 7 days, be the results are shown in
fig. 2.
Result shows, and the cultural method cultured cells of deciduous teeth dental pulp stem cell of the present invention is at original cuiture after 7 days, and cell confluency degree can reach more than 80%, and in comparative example, cell confluency degree, about 50%, is starkly lower than cultural method of the present invention.
Embodiment 4, cell expansion are cultivated
With application number, disclosed in 201210514497.X, method cultured cells is as a comparison case (control group), according to the method culturing cell of embodiment 1, when Growth of Cells converges to 80%-90%, 0.125% trypsin digestion and cell, the centrifugal 3-5min of 1000rpm-2000rpm, collecting cell precipitates, with Lonza perfect medium re-suspended cell, and get 20 μ L cell suspensions and carry out cell counting, count to obtain primary cell total amount, and to adjust cell density be 2 × 10
3/ cm
2-5 × 10
3/ cm
2, be inoculated in 6 orifice plate Tissue Culture Dishs.Cultivate with 20mlLonza perfect medium, then add Urogastron EGF and Basic Fibroblast Growth Factor bFGF (both final concentrations are 10ng/mL), mix cell suspension, be put in 37 DEG C, humidity is cultivate in the CO2gas incubator of 95%.When P1 converges to 80%-90% for Growth of Cells, according to above-mentioned identical collection step cell, and P1 is counted for cell.Result
as Fig. 3.
By
fig. 3result is visible, and 1 × 10
5primary cell goes down to posterity, and cultural method (sample group) cultured cells of deciduous teeth dental pulp stem cell of the present invention has increased nearly 20 times, and in control group, total cellular score has only increased about 6 times.
Embodiment 5, cell surface marker quality testing are surveyed
Respectively the method for Example 1 cultivate the 1st, 6,10 generation cell, with the cell surface marker between the different algebraically of flow cytomery.Digestion collecting cell, after counting, often pipe adds 2 × 10
5cell count, dye solution washes 1 time, the centrifugal 5min of 1000rpm; Abandon supernatant, with dye solution piping and druming mixing cell; Add each 2 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 1000rpm; The cell of direct mark abandons supernatant, and lucifuge adds the sample-loading buffer of 500 μ L, and mixing, with 200 eye mesh screen filtration cell samples, flow cytomery cell-surface antigens, the results are shown in
fig. 4,
fig. 5with
fig. 6.
By
fig. 4-6 results are visible, the surface marker of flow cytomery cell, observe 1st generation, the 6th generation, the 10th generation the surface marker of cell obviously do not change.Deciduous teeth dental pulp stem cell surface marker CD45 (white corpuscle is positive), HLA-DR (MHC-II quasi-molecule) are for all to present feminine gender, and deciduous teeth dental pulp stem cell surface marker CD59, CD90 all present the positive simultaneously.The later deciduous teeth dental pulp stem cell cellular form of the third generation is homogeneous, and purity is more than 98%.
Embodiment 6, P3 become fat to break up qualification for cell induction
Method is: when cell cultures is to 2nd generation, according to 1 × 10
3cell/cm
2be inoculated in six orifice plates, add perfect medium, after 24h, add adipogenic induction substratum and cultivate.Adipogenic induction substratum comprises basic medium DMEM, 10% foetal calf serum, 0.5mMIBMX, 1 μM of dexamethasone, 100 μMs of indomethacins, 5 μ g/ml Regular Insulin, 2mm/L glutamine etc.Within every three days, change liquid.Carry out oil red O stain after surrounding, qualification fat drips formational situation, the results are shown in
fig. 7.
fig. 7result shows, and after cultivating surrounding, the known control group of Microscopic observation, without induction, does not occur that fat drips, and experimental group, namely cultural method cultured cells described in embodiment 1 is after induction, carries out oil red O stain, and visible significantly fat drips formation.
Embodiment 7, P3 identify for cell induction Osteoblast Differentiation
Method is: when cell cultures is to 2nd generation, according to 1 × 10
3cell/cm
2be inoculated in six orifice plates, add perfect medium, after 24h, add after adipogenic induction substratum carries out cultivation 24h, add Osteogenic Induction Medium and carry out inducing culture, changed liquid every 2-3 days, carry out VonKossa dyeing after 21 days, the results are shown in
fig. 8.
fig. 8result shows, and compared with control group, induction group has obvious calcium tubercle, and this is the feature that scleroblast is formed.
Claims (10)
1. the cultural method of a deciduous teeth dental pulp stem cell, get pulp tissue, add type i collagen enzyme after shredding, 37 DEG C, 200rpm digests 10-20min, serum free medium stops digestion, piping and druming discrete cellular agglomerate obtains single discrete cell, adds cell suspension re-suspended cell and adjusts cell density, in 37 DEG C, humidity is cultivate in the CO2gas incubator of 95%, when Growth of Cells merges to 80%-90%, use containing Secondary Culture after tryptic Digestive system peptic cell; Wherein said cell suspension is made up of serum free medium, Urogastron, Basic Fibroblast Growth Factor.
2. cultural method according to claim 1, also comprises the step of cleaning single discrete cellular.
3. cultural method according to claim 2, described cleaning is specially centrifugal rear PBS washing and precipitating, centrifugal collecting precipitation.
4. the cultural method according to claim 1-3 any one, diameter≤70 μm of described single discrete cell.
5. the cultural method according to claim 1-4 any one, the add-on of described type i collagen enzyme is 10-20 times of volume of pulp tissue's volume.
6. the cultural method according to claim 1-5 any one, described cell density is 2 × 10
3/cm
2-5 × 10
3/ cm
2.
7. the cultural method according to claim 1-6 any one, the final concentration of Urogastron described in described cell suspension is 10ng/mL.
8. the cultural method according to claim 1-7 any one, the final concentration of Basic Fibroblast Growth Factor described in described cell suspension is 10ng/mL.
9. the cultural method according to claim 1-8 any one, changed a cell suspension every three days in described culturing process.
10. the cultural method according to claim 1-9 any one, described tryptic concentration is 0.125%.
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| CN105754934A (en) * | 2016-03-08 | 2016-07-13 | 大连大学 | Dental pulp stem cell, preparation method thereof and related bone tissue engineering material |
| CN106539821A (en) * | 2016-11-30 | 2017-03-29 | 广州赛莱拉干细胞科技股份有限公司 | A kind of stem cell excretion body preparation and its preparation method and application |
| CN107227295A (en) * | 2016-03-23 | 2017-10-03 | 北京泰盛生物科技有限公司 | Come off separation and the in-vitro multiplication method of deciduous teeth stem cell |
| CN110898077A (en) * | 2019-11-20 | 2020-03-24 | 青海晨菲制药有限公司 | Stem cell composition and application thereof |
| CN110946821A (en) * | 2019-11-29 | 2020-04-03 | 上海长一干细胞研究中心有限公司 | High-moisturizing skin-moistening and reconstructing children deciduous tooth pulp stem cell mask |
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| CN111172107A (en) * | 2020-03-02 | 2020-05-19 | 上海市口腔病防治院 | Dental pulp stem cell conditioned medium and preparation method and application thereof |
| CN111548990A (en) * | 2020-05-22 | 2020-08-18 | 深圳至博生物科技有限公司 | Dental pulp stem cell polymer for exfoliating deciduous teeth and preparation method and application thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105754934A (en) * | 2016-03-08 | 2016-07-13 | 大连大学 | Dental pulp stem cell, preparation method thereof and related bone tissue engineering material |
| CN105754934B (en) * | 2016-03-08 | 2019-07-12 | 大连大学 | Dental pulp stem cell, preparation method and its related engineering material of bone tissue |
| CN107227295A (en) * | 2016-03-23 | 2017-10-03 | 北京泰盛生物科技有限公司 | Come off separation and the in-vitro multiplication method of deciduous teeth stem cell |
| CN106539821A (en) * | 2016-11-30 | 2017-03-29 | 广州赛莱拉干细胞科技股份有限公司 | A kind of stem cell excretion body preparation and its preparation method and application |
| CN110898077A (en) * | 2019-11-20 | 2020-03-24 | 青海晨菲制药有限公司 | Stem cell composition and application thereof |
| CN110946821A (en) * | 2019-11-29 | 2020-04-03 | 上海长一干细胞研究中心有限公司 | High-moisturizing skin-moistening and reconstructing children deciduous tooth pulp stem cell mask |
| CN110951683A (en) * | 2020-01-06 | 2020-04-03 | 深圳华云生物科技发展有限公司 | Preparation method of dental pulp stem cells |
| CN111172107A (en) * | 2020-03-02 | 2020-05-19 | 上海市口腔病防治院 | Dental pulp stem cell conditioned medium and preparation method and application thereof |
| CN111172107B (en) * | 2020-03-02 | 2023-09-19 | 上海市口腔病防治院 | Dental pulp stem cell conditioned medium and preparation method and application thereof |
| CN111548990A (en) * | 2020-05-22 | 2020-08-18 | 深圳至博生物科技有限公司 | Dental pulp stem cell polymer for exfoliating deciduous teeth and preparation method and application thereof |
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Application publication date: 20151125 |