CN113604430A - Method for culturing dental pulp mesenchymal stem cells - Google Patents
Method for culturing dental pulp mesenchymal stem cells Download PDFInfo
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- CN113604430A CN113604430A CN202111038934.0A CN202111038934A CN113604430A CN 113604430 A CN113604430 A CN 113604430A CN 202111038934 A CN202111038934 A CN 202111038934A CN 113604430 A CN113604430 A CN 113604430A
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- 210000003074 dental pulp Anatomy 0.000 title claims abstract description 43
- 238000012258 culturing Methods 0.000 title claims abstract description 24
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000005070 sampling Methods 0.000 claims abstract description 28
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 25
- 210000004262 dental pulp cavity Anatomy 0.000 claims abstract description 19
- 239000012530 fluid Substances 0.000 claims abstract description 18
- 210000004268 dentin Anatomy 0.000 claims abstract description 14
- 239000001963 growth medium Substances 0.000 claims abstract description 12
- 102000029816 Collagenase Human genes 0.000 claims abstract description 10
- 108060005980 Collagenase Proteins 0.000 claims abstract description 10
- 229960002424 collagenase Drugs 0.000 claims abstract description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 9
- 239000006143 cell culture medium Substances 0.000 claims abstract description 7
- 230000029087 digestion Effects 0.000 claims abstract description 7
- 238000011010 flushing procedure Methods 0.000 claims abstract description 7
- 239000007787 solid Substances 0.000 claims abstract description 5
- 230000003321 amplification Effects 0.000 claims abstract description 4
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- 238000001914 filtration Methods 0.000 claims abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 238000002791 soaking Methods 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 4
- 210000000130 stem cell Anatomy 0.000 claims description 12
- 238000005553 drilling Methods 0.000 claims description 8
- 239000000645 desinfectant Substances 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000003973 irrigation Methods 0.000 claims description 3
- 230000002262 irrigation Effects 0.000 claims description 3
- 230000003902 lesion Effects 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000010298 pulverizing process Methods 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000011278 co-treatment Methods 0.000 claims description 2
- 210000003298 dental enamel Anatomy 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 abstract description 4
- 239000012535 impurity Substances 0.000 abstract description 4
- 210000005258 dental pulp stem cell Anatomy 0.000 abstract description 3
- 210000005036 nerve Anatomy 0.000 abstract description 3
- 239000000835 fiber Substances 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- 238000003860 storage Methods 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 16
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000001089 mineralizing effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
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- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The invention discloses a method for culturing dental pulp mesenchymal stem cells, which comprises the steps of cleaning fallen teeth, putting the cleaned teeth into a sterile sampling bottle, and putting the sterile sampling bottle into a medical ice bag for storage; flushing the dental pulp cavity with normal saline to make the pulp be carried out of the dental pulp cavity by water flow and enter the normal saline; filtering physiological saline with pulp to obtain solid pulp, and soaking the pulp in physiological saline to obtain pulp tissue liquid; collagenase is added into the dental pulp tissue fluid, the dental pulp tissue fluid is digested for 30 minutes, then the dental pulp stem cell culture medium after the digestion is stopped is resuspended, and the culture medium is inoculated into a culture flask for amplification culture. According to the invention, the dentin is drilled through during sampling, then the sampling is flushed, so that the enamel is prevented from being polluted during sampling, impurities of a dentin cell layer, a cell-poor layer and a multicellular layer are prevented from being taken during sampling, and the dental pulp tissue fluid obtained by crushing the enamel and then immersing the enamel in normal saline can reduce impurity components such as fibers and nerves in dental pulp.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a method for culturing dental pulp mesenchymal stem cells.
Background
The pulp tissue is located within the pulp chamber inside the tooth and is the only soft tissue in the tooth tissue. Fibroblast cells separated from dental pulp tissues are called dental pulp stem cells, the dental pulp stem cells have the potential of multidirectional differentiation, can form cells with the capability of mineralizing nodules, and can also be differentiated into cell line types such as fat, bone, cartilage, muscle, vascular endothelium, liver, nerve and the like through the induction of different cytokines.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a method for culturing dental pulp mesenchymal stem cells.
The invention realizes the purpose through the following technical scheme: a method for culturing dental pulp mesenchymal stem cells comprises the following steps:
(1) tooth preservation: cleaning the fallen teeth and putting the teeth into a sterile sampling bottle;
(2) sampling pulp quality: taking out the tooth from the sampling bottle, enabling the drill to penetrate through dentin to expose dental pulp, and then flushing the dental pulp cavity by using normal saline to enable the dental pulp to be taken out of the dental pulp cavity by water flow and enter the normal saline;
(3) sample treatment: filtering physiological saline with pulp to obtain solid pulp, pulverizing the pulp, and soaking the pulverized pulp in physiological saline to obtain pulp tissue fluid;
(4) extracting stem cells: adding collagenase into the dental pulp tissue fluid, transferring the dental pulp tissue fluid into a centrifuge tube for digestion for 30 minutes, adding the digested tissue fluid into a culture medium to terminate digestion, centrifuging the culture medium, resuspending the pulp stem cell culture medium, and inoculating the pulp stem cell culture medium into a culture bottle;
(5) amplification culture: transferring the culture medium in the culture flask in the step (4) to a twelve-well plate, and performing 4.5% -5.5% CO treatment at 35-37 deg.C2And culturing under saturated humidity condition.
Preferably: the tooth in the step (1) is a healthy tooth without a pulp tissue lesion.
Preferably: when the teeth are cleaned in the step (1), the surfaces of the teeth are firstly wiped by medical disinfectant, and then the teeth are soaked in physiological saline to dilute the disinfectant on the surfaces.
Preferably: after the tooth surface is wiped in the step (1), standing for more than 60 seconds, and then immersing the tooth into physiological saline.
Preferably: when sampling is performed in the step (2), holes are drilled from both sides of the tooth, and the drilling of each side is stopped after the drill bit penetrates dentin, so that the dentin is exposed.
Preferably: before drilling in the step (2), the teeth need to be wiped dry and fixed.
Preferably: when the pulp chamber is flushed with the physiological saline in the step (2), the pulp chamber needs to be flushed under pressure, and the physiological saline for flushing the pulp chamber is recycled.
Preferably: when the irrigation is performed in the step (2), the physiological saline enters the pulp cavity from the hole on one side of the tooth and flows out of the pulp cavity from the hole on the other side.
Preferably: the step (2) is carried out in a sterile environment when sampling is carried out.
Preferably: the collagenase in the step (4) is collagenase type I with the concentration of 10mg/ml-12 mg/ml.
Preferably: and (4) pouring the digested tissue fluid into a culture medium in the step (4), and slowly and uniformly stirring.
Preferably: the upper connecting rod is hinged with the enclosure, and the lower connecting rod is hinged with the enclosure.
Preferably: the temperature at which the centrifugation in the step (4) is performed is 25 ℃, the time is 10 minutes, and the centrifugal force is 1100rpm/min to 1400 rpm/min.
Preferably: and (5) after the dental pulp mesenchymal stem cells are subjected to expansion culture for 12 hours, sampling and observing through a microscope, and screening the stem cells.
Compared with the prior art, the invention has the beneficial effects that:
1. drilling through dentin at the time of sampling and then rinsing the sample can avoid contamination of enamel at the time of sampling, and rinsing can avoid taking impurities of a dentin cell layer, a cell-poor layer and a multicellular layer at the time of sampling;
2. dental pulp tissue liquid obtained by crushing dental enamel and immersing the same in physiological saline can reduce impurity components such as fibers and nerves in dental pulp.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
Fig. 1 is a schematic diagram of the steps of a method for culturing dental pulp mesenchymal stem cells according to the present invention.
Detailed Description
In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on those shown in the drawings, and are used only for convenience in describing the present invention and for simplicity in description, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be construed as limiting the present invention. Furthermore, the terms "first", "second", etc. are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first," "second," etc. may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless otherwise specified.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art through specific situations.
The invention will be further described with reference to the accompanying drawings in which:
example 1
As shown in fig. 1, a method for culturing dental pulp mesenchymal stem cells comprises the following steps:
(1) tooth preservation: cleaning the fallen teeth, putting the cleaned teeth into a sterile sampling bottle, and then putting the sterile sampling bottle into a medical ice bag for storage for 24 hours to sample the tooth medulla;
(2) sampling pulp quality: taking out the tooth from the sampling bottle, enabling the drill to penetrate through dentin to expose dental pulp, and then flushing the dental pulp cavity by using normal saline to enable the dental pulp to be taken out of the dental pulp cavity by water flow and enter the normal saline;
(3) sample treatment: filtering physiological saline with pulp to obtain solid pulp, pulverizing the pulp, and soaking the pulverized pulp in physiological saline to obtain pulp tissue fluid;
(4) extracting stem cells: adding collagenase into the dental pulp tissue fluid, transferring the dental pulp tissue fluid into a centrifuge tube for digestion for 30 minutes, adding the digested tissue fluid into a culture medium to terminate digestion, centrifuging the culture medium, resuspending the pulp stem cell culture medium, and inoculating the pulp stem cell culture medium into a culture bottle;
(5) amplification culture: transferring the medium from the culture flask of step (4) to a twelve-well plate at 36.5 ℃ with 5.2% CO2And culturing under saturated humidity condition.
The tooth in the step (1) is a healthy tooth without a pulp tissue lesion. When the teeth are cleaned in the step (1), the surfaces of the teeth are firstly wiped by medical disinfectant, and then the teeth are soaked in physiological saline to dilute the disinfectant on the surfaces. After the tooth surface is wiped in the step (1), standing for more than 60 seconds, and then immersing the tooth into physiological saline. When sampling is performed in the step (2), holes are drilled from both sides of the tooth, and the drilling of each side is stopped after the drill bit penetrates dentin, so that the dentin is exposed. Before drilling in the step (2), the teeth need to be wiped dry and fixed. When the pulp chamber is flushed with the physiological saline in the step (2), the pulp chamber needs to be flushed under pressure, and the physiological saline for flushing the pulp chamber is recycled. When the irrigation is performed in the step (2), the physiological saline enters the pulp cavity from the hole on one side of the tooth and flows out of the pulp cavity from the hole on the other side. The step (2) is carried out in a sterile environment when sampling is carried out. The collagenase in the step (4) is collagenase type I with the concentration of 10mg/ml-12 mg/ml. And (4) pouring the digested tissue fluid into a culture medium in the step (4), and slowly and uniformly stirring. The upper connecting rod is hinged with the enclosure, and the lower connecting rod is hinged with the enclosure. The temperature at which the centrifugation in the step (4) is performed is 25 ℃, the time is 10 minutes, and the centrifugal force is 1100rpm/min to 1400 rpm/min. And (5) after the dental pulp mesenchymal stem cells are subjected to expansion culture for 12 hours, sampling and observing through a microscope, and screening the stem cells.
Example 2
Example 2 differs from example 1 in that: in drilling through the dentin in step (4), the drill is driven from both the front and back sides of the tooth, thus allowing the pulp to be washed from the largest cross-section of the pulp chamber.
Example 3
Example 3 differs from example 1 in that: when the pulp is washed in the step (2), the pulp is washed again after the physiological saline is filtered, so that the washing device can be prevented from being clogged by the solid pulp.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Claims (13)
1. A method for culturing dental pulp mesenchymal stem cells is characterized in that: the method comprises the following steps:
(1) tooth preservation: cleaning the fallen teeth and storing in a sterile sampling bottle;
(2) sampling pulp quality: taking out the tooth from the sampling bottle, enabling the drill to penetrate through dentin to expose dental pulp, and then flushing the dental pulp cavity by using normal saline to enable the dental pulp to be taken out of the dental pulp cavity by water flow and enter the normal saline;
(3) sample treatment: filtering physiological saline with pulp to obtain solid pulp, pulverizing the pulp, and soaking the pulverized pulp in physiological saline to obtain pulp tissue fluid;
(4) extracting stem cells: adding collagenase into the dental pulp tissue fluid, transferring the dental pulp tissue fluid into a centrifuge tube for digestion for 30 minutes, adding the digested tissue fluid into a culture medium to terminate digestion, centrifuging the culture medium, resuspending the pulp stem cell culture medium, and inoculating the pulp stem cell culture medium into a culture bottle;
(5) amplification culture: transferring the culture medium in the culture flask in the step (4) to a twelve-well plate, and performing 4.5% -5.5% CO treatment at 35-37 deg.C2And culturing under saturated humidity condition.
2. The method for culturing dental pulp mesenchymal stem cells according to claim 1, wherein: the tooth in the step (1) is a healthy tooth without a pulp tissue lesion.
3. The method for culturing dental pulp mesenchymal stem cells according to claim 1, wherein: when the teeth are cleaned in the step (1), the surfaces of the teeth are firstly wiped by medical disinfectant, and then the teeth are soaked in physiological saline to dilute the disinfectant on the surfaces.
4. The method for culturing dental pulp mesenchymal stem cells according to claim 3, wherein: after the tooth surface is wiped in the step (1), standing for more than 60 seconds, and then immersing the tooth into physiological saline.
5. The method for culturing dental pulp mesenchymal stem cells according to claim 1, wherein: when sampling is performed in the step (2), holes are drilled from both sides of the tooth, and the drilling of each side is stopped after the drill bit penetrates dentin, so that the dentin is exposed.
6. The method for culturing dental pulp mesenchymal stem cells according to claim 4, wherein: before drilling in the step (2), the teeth need to be wiped dry and fixed.
7. The method for culturing dental pulp mesenchymal stem cells according to claim 1, wherein: when the pulp chamber is flushed with the physiological saline in the step (2), the pulp chamber needs to be flushed under pressure, and the physiological saline for flushing the pulp chamber is recycled.
8. The method for culturing dental pulp mesenchymal stem cells according to claim 6, wherein: when the irrigation is performed in the step (2), the physiological saline enters the pulp cavity from the hole on one side of the tooth and flows out of the pulp cavity from the hole on the other side.
9. The method for culturing dental pulp mesenchymal stem cells according to claim 1, wherein: the step (2) is carried out in a sterile environment when sampling is carried out.
10. The method for culturing dental pulp mesenchymal stem cells according to claim 1, wherein: the collagenase in the step (4) is collagenase type I with the concentration of 10mg/ml-12 mg/ml.
11. The method for culturing dental pulp mesenchymal stem cells according to claim 1, wherein: and (4) pouring the digested tissue fluid into a culture medium in the step (4), and slowly and uniformly stirring.
12. The method for culturing dental pulp mesenchymal stem cells according to claim 1, wherein: the temperature at which the centrifugation in the step (4) is performed is 25 ℃, the time is 10 minutes, and the centrifugal force is 1100rpm/min to 1400 rpm/min.
13. The method for culturing dental pulp mesenchymal stem cells according to claim 1, wherein: and (5) after the dental pulp mesenchymal stem cells are subjected to expansion culture for 12 hours, sampling and observing through a microscope, and screening the stem cells.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111038934.0A CN113604430A (en) | 2021-09-06 | 2021-09-06 | Method for culturing dental pulp mesenchymal stem cells |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111038934.0A CN113604430A (en) | 2021-09-06 | 2021-09-06 | Method for culturing dental pulp mesenchymal stem cells |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105802905A (en) * | 2016-04-21 | 2016-07-27 | 天津普瑞赛尔生物科技有限公司 | Kit for collecting, separating and amplifying dental pulp stem cells and using method thereof |
| WO2017058003A2 (en) * | 2015-10-02 | 2017-04-06 | Elixir Biopharma Sdn. Bhd. | Method of isolating mesenchymal stromal cells and applications for tissue engineering |
| CN108277202A (en) * | 2018-01-19 | 2018-07-13 | 深圳中生健康管理有限公司 | A kind of cultural method of dental pulp mescenchymal stem cell |
| JP2018201340A (en) * | 2017-05-30 | 2018-12-27 | 東洋紡株式会社 | Method for storing dental pulp tissue and method for culturing stem cells from stored dental pulp tissue |
| CN111334465A (en) * | 2019-12-30 | 2020-06-26 | 上海循益生物技术有限公司 | Preparation method of dental pulp mesenchymal stem cells |
-
2021
- 2021-09-06 CN CN202111038934.0A patent/CN113604430A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017058003A2 (en) * | 2015-10-02 | 2017-04-06 | Elixir Biopharma Sdn. Bhd. | Method of isolating mesenchymal stromal cells and applications for tissue engineering |
| CN105802905A (en) * | 2016-04-21 | 2016-07-27 | 天津普瑞赛尔生物科技有限公司 | Kit for collecting, separating and amplifying dental pulp stem cells and using method thereof |
| JP2018201340A (en) * | 2017-05-30 | 2018-12-27 | 東洋紡株式会社 | Method for storing dental pulp tissue and method for culturing stem cells from stored dental pulp tissue |
| CN108277202A (en) * | 2018-01-19 | 2018-07-13 | 深圳中生健康管理有限公司 | A kind of cultural method of dental pulp mescenchymal stem cell |
| CN111334465A (en) * | 2019-12-30 | 2020-06-26 | 上海循益生物技术有限公司 | Preparation method of dental pulp mesenchymal stem cells |
Non-Patent Citations (2)
| Title |
|---|
| 李远;关方霞;李季;杨波;胡祥;杜英;谷晨熙;雷宁静;王晓薇;: "改良法培养人牙髓间充质干细胞及其向神经样细胞的分化", 中国组织工程研究与临床康复 * |
| 李颖;谷子芽;吕秋峰;尤金彪;刘芳菲;: "酶解组织块法原代培养人牙周膜干细胞和牙髓干细胞", 中国组织工程研究 * |
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