CN106754658A - A kind of culture medium for cultivating dental pulp stem cell and preparation method thereof - Google Patents
A kind of culture medium for cultivating dental pulp stem cell and preparation method thereof Download PDFInfo
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- CN106754658A CN106754658A CN201611199904.7A CN201611199904A CN106754658A CN 106754658 A CN106754658 A CN 106754658A CN 201611199904 A CN201611199904 A CN 201611199904A CN 106754658 A CN106754658 A CN 106754658A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention belongs to technical field of cell culture, and in particular to a kind of culture medium of culture dental pulp stem cell and preparation method thereof.The culture medium of present invention culture dental pulp stem cell, including additive in the basal medium of basal medium and addition, the composition of the additive with final concentration, including：The μ g/L of EGF 35; the 4g/L of carboxymethyl chitosan 2, the 8mg/L of catalase 5, the 0.18mg/L of lipoic acid 0.14; 0.3 0.6 μM of Co-Q10; the μ g/L of LN1 5 25,25 μM of leukotrienes, 8 14 μM of niacinamide; the 4mg/L of taurine 2; the μ g/L of platelet derived growth factor 26,70 80 μM of Choline Chloride, the 2mg/L of diosgenin 0.5.The culture medium price of present invention culture dental pulp stem cell is relatively low, safe, can significantly improve the amplification rate of pulp cells.
Description
Technical field
The invention belongs to technical field of cell culture, and in particular to a kind of culture medium of culture dental pulp stem cell and its preparation
Method.
Background technology
Due to defect of teeth and absence of tooth caused by a variety of causes, it has also become harm oral cavity so that health it is main
Disease, its incidence of disease increases year by year.At present, tooth defect disease mainly is tackled by means of the filling of various dental materials, though
So the continuation of lesion can be prevented to develop to a certain extent, but root problem can not be solved.For absence of tooth although solving
Approach is a lot, but most effective and most basic method or the biological regeneration of tooth.
It is that effective treatment of defect of teeth and absence of tooth brings hope with the fast development of regeneration medical science.
The interaction of existing many research and utilization Dental epitheliums and Odontogenic cysts mesenchyma at present has regenerated dental tissue.Conventional
Odontogenic cysts mesenchyma includes dental papilla cells and Dental Pulp Cells.Clinically it is difficult to obtain due to dental papilla cells, therefore,
Currently used for tooth body and regeneration of tooth seed cell first-selection be pulp tissue source cell, most important of which is that dental pulp is dry
Cell.
Chinese patent application CN104711219A discloses a kind of dental pulp stem cell culture medium, and basal medium is IMDM,
Contain SITE100* in 1L culture mediums, ascorbic acid 200-400mM, SITE be cell growth necessary to component, ascorbic acid and
Fibronectin contributes to dental pulp stem cell to form extracellular matrix, beneficial to dental pulp stem cell growth, contains PDGF1-10ug, hydrogenation
Cortisone 1-10ug, EGF1-5ng, b-FGF1-5ng, PTH50-200ng, dexamethasone 1-20mM.
The premise of Clinical practice dental pulp stem cell is that substantial amounts of amplification is carried out to dental pulp stem cell, and existing amplification method is most
Conventional is the culture medium using 10% hyclone of addition, and culture medium of the Clinical practice containing animal sources can not only cause immune row
Reprimand reaction, heterologus virus infection, and also using the dental pulp stem cell of this medium culture, growth fraction is slower, and it is super in passage
The potential for crossing 5 its differentiation function cells afterwards can be substantially reduced.Although having been developed for the culture dental pulp without serum dry thin
The culture medium of born of the same parents, but it is expensive.
The content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to provide it is a kind of cultivate dental pulp stem cell culture medium and its
Preparation method.The medium component of the culture dental pulp stem cell that the present invention is provided is clear and definite and price is relatively low, does not contain hyclone
And without any animal origin composition, it is safe, the amplification rate of pulp cells can be significantly improved, and do not influence dental pulp to do
The Multidirectional Differentiation ability of cell.
The technical scheme is that：
A kind of culture medium for cultivating dental pulp stem cell, including basal medium and addition adding in the basal medium
Plus agent, the composition of the additive with final concentration, including：EGF 3-5 μ g/L, carboxymethyl chitosan 2-4g/L,
Catalase 5-8mg/L, lipoic acid 0.14-0.18mg/L, 0.3-0.6 μM of Co-Q10, LN1 5-25 μ g/L,
2-5 μM of leukotrienes, 8-14 μM of niacinamide, taurine 2-4mg/L, platelet derived growth factor 2-6 μ g/L, Choline Chloride 70-
80 μM, diosgenin 0.5-2mg/L.
A kind of culture medium for cultivating dental pulp stem cell, preferred scheme is, including basal medium and addition are in the base
Additive in basal culture medium, the composition of the additive with final concentration, including：The μ g/L of EGF 4, carboxymethyl shell
Glycan 3g/L, catalase 6mg/L, lipoic acid 0.16mg/L, 0.4 μM of Co-Q10, the μ g/L of LN1 8, leukotrienes
4 μM, 12 μM of niacinamide, taurine 3mg/L, the μ g/L of platelet derived growth factor 5,76 μM of Choline Chloride, diosgenin
1.4mg/L。
The basal medium is DMEM or F12 culture mediums.
In addition, present invention also offers the preparation method of the culture medium for cultivating dental pulp stem cell, step is as follows：
S1 is to addition EGF, lipoic acid, laminin, leukotrienes, niacinamide, ox sulphur in basal medium
Acid, platelet derived growth factor and Choline Chloride, stir 20-40 minutes, add carboxymethyl chitosan and diosgenin, after
Continuous stirring 30-40 minutes, stands 1-3 hours, adds catalase and Co-Q10, stirs 30 minutes, obtains mixture；
The pH to 6.8-7.1 of S2 regulating steps S1 gained mixtures, with 0.2-0.25 micron membrane filter filtration sterilizations, obtains final product.
Preferably, the step S1 is stirred 30 minutes.
Preferably, the step S1 continues to stir 35 minutes.
Preferably, the step S1 stands 2 hours.
Preferably, the pH to 7.0 of the step S1 regulating steps S2 gained mixture.
Preferably, the step S2 is with 0.23 micron membrane filter filtration sterilization.
The culture medium of present invention culture dental pulp stem cell, including basal medium and addition are in the basal medium
Additive, the additive of addition is including EGF, carboxymethyl chitosan, Choline Chloride and diosgenin etc..This hair
Each composition synergy, can significantly improve the amplification rate of dental pulp stem cell in the culture medium of bright culture dental pulp stem cell, and
The Multidirectional Differentiation ability of dental pulp stem cell is not influenceed.
Compared with prior art, the culture medium of the culture dental pulp stem cell that the present invention is provided has the advantage that：
(1) medium component of the culture dental pulp stem cell that the present invention is provided is clear and definite and price is relatively low.
(2) culture medium of the culture dental pulp stem cell that the present invention is provided does not contain hyclone and without any animal origin
Composition, it is safe.
(3) culture medium of the culture dental pulp stem cell that the present invention is provided can significantly improve the amplification rate of pulp cells,
And do not influence the Multidirectional Differentiation ability of dental pulp stem cell.
Specific embodiment
Below by way of the description of specific embodiment, the invention will be further described, but this is not to limit of the invention
System, those skilled in the art's basic thought of the invention, various modifications may be made or improves, but without departing from this
The basic thought of invention, within the scope of the present invention.
Carboxymethyl chitosan used of the invention is purchased from ZHEJIANG AOXING BIOTECHNOLOGY CO., LTD, and DMEM culture mediums are purchased from
The vast Tyke biological gene technology Co., Ltd in Beijing, F12 culture mediums are purchased from the vast Tyke biological gene technology in Beijing
Co., Ltd, diosgenin is purchased from Xi'an Tong Ze bio tech ltd.
Embodiment 1, a kind of culture medium for cultivating dental pulp stem cell
The culture medium of the culture dental pulp stem cell, including basal medium and addition adding in the basal medium
Plus agent, the composition of the additive with final concentration, including：EGF 3 μ g/L, carboxymethyl chitosan 2g/L, peroxide
Change hydrogen enzyme 5mg/L, lipoic acid 0.14mg/L, 0.3 μM of Co-Q10, the μ g/L of LN1 5,2 μM of leukotrienes, the μ of niacinamide 8
M, taurine 2mg/L, the μ g/L of platelet derived growth factor 2,70 μM of Choline Chloride, diosgenin 0.5mg/L.
The basal medium is F12 culture mediums.
Preparation method：
S1 is to addition EGF, lipoic acid, laminin, leukotrienes, niacinamide, ox sulphur in basal medium
Acid, platelet derived growth factor and Choline Chloride, stir 20 minutes, add carboxymethyl chitosan and diosgenin, continue
Stirring 30 minutes, stands 1 hour, adds catalase and Co-Q10, stirs 30 minutes, obtains mixture；
The pH to 6.8 of S2 regulating steps S1 gained mixtures, with 0.2 micron membrane filter filtration sterilization, obtains final product.
Embodiment 2, a kind of culture medium for cultivating dental pulp stem cell
The culture medium of the culture dental pulp stem cell, including basal medium and addition adding in the basal medium
Plus agent, the composition of the additive with final concentration, including：EGF 5 μ g/L, carboxymethyl chitosan 4g/L, peroxide
Change hydrogen enzyme 8mg/L, lipoic acid 0.18mg/L, 0.6 μM of Co-Q10, the μ g/L of laminin 25,5 μM of leukotrienes, the μ of niacinamide 14
M, taurine 4mg/L, the μ g/L of platelet derived growth factor 6,80 μM of Choline Chloride, diosgenin 2mg/L.
The basal medium is DMEM culture mediums.
Preparation method：
S1 is to addition EGF, lipoic acid, laminin, leukotrienes, niacinamide, ox sulphur in basal medium
Acid, platelet derived growth factor and Choline Chloride, stir 40 minutes, add carboxymethyl chitosan and diosgenin, continue
Stirring 40 minutes, stands 3 hours, adds catalase and Co-Q10, stirs 30 minutes, obtains mixture；
The pH to 7.1 of S2 regulating steps S1 gained mixtures, with 0.25 micron membrane filter filtration sterilization, obtains final product.
Embodiment 3, a kind of culture medium for cultivating dental pulp stem cell
The culture medium of the culture dental pulp stem cell, including basal medium and addition adding in the basal medium
Plus agent, the composition of the additive with final concentration, including：EGF 4 μ g/L, carboxymethyl chitosan 3g/L, peroxide
Change hydrogen enzyme 6mg/L, lipoic acid 0.16mg/L, 0.4 μM of Co-Q10, the μ g/L of LN1 8,4 μM of leukotrienes, the μ of niacinamide 12
M, taurine 3mg/L, the μ g/L of platelet derived growth factor 5,76 μM of Choline Chloride, diosgenin 1.4mg/L.
The basal medium is F12 culture mediums.
Preparation method：
S1 is to addition EGF, lipoic acid, laminin, leukotrienes, niacinamide, ox sulphur in basal medium
Acid, platelet derived growth factor and Choline Chloride, stir 30 minutes, add carboxymethyl chitosan and diosgenin, continue
Stirring 35 minutes, stands 2 hours, adds catalase and Co-Q10, stirs 30 minutes, obtains mixture；
The pH to 7.0 of S2 regulating steps S1 gained mixtures, with 0.23 micron membrane filter filtration sterilization, obtains final product.
Comparative example 1, a kind of culture medium for cultivating dental pulp stem cell
The culture medium of the culture dental pulp stem cell, including basal medium and addition adding in the basal medium
Plus agent, the composition of the additive with final concentration, including：EGF 4 μ g/L, water soluble chitosan 3g/L, peroxide
Change hydrogen enzyme 6mg/L, lipoic acid 0.16mg/L, 0.4 μM of Co-Q10, the μ g/L of LN1 8,4 μM of leukotrienes, the μ of niacinamide 12
M, taurine 3mg/L, the μ g/L of platelet derived growth factor 5,76 μM of Choline Chloride, diosgenin 1.4mg/L.
The basal medium is F12 culture mediums.
Preparation method is similar to Example 3.
Difference with embodiment 3 is that carboxymethyl chitosan is replaced with into water soluble chitosan.
Comparative example 2, a kind of culture medium for cultivating dental pulp stem cell
The culture medium of the culture dental pulp stem cell, including basal medium and addition adding in the basal medium
Plus agent, the composition of the additive with final concentration, including：EGF 4 μ g/L, carboxymethyl chitosan 3g/L, peroxide
Change hydrogen enzyme 6mg/L, lipoic acid 0.16mg/L, 0.4 μM of Co-Q10, the μ g/L of LN1 8,4 μM of leukotrienes, the μ of niacinamide 12
M, taurine 3mg/L, the μ g/L of platelet derived growth factor 5,76 μM of Choline Chloride, Cucurbitacin B 1.4mg/L.
The basal medium is F12 culture mediums.
Preparation method is similar to Example 3.
Difference with embodiment 3 is that diosgenin is replaced with into Cucurbitacin B.
Test example one, the culture medium of present invention culture dental pulp stem cell are to dental pulp stem cell culture effect
1st, tested culture medium：The culture medium of embodiment of the present invention 1-3 gained culture dental pulp stem cells, and the He of comparative example 1
The culture medium of the gained culture dental pulp stem cell of comparative example 2.
2nd, source of human stem cell is cultivated：From the dental pulp stem cell of dental pulp.
3rd, cell culture experiments in vitro
Pulp tissue, the pulp tissue of excision root tip 1mm are gripped with aseptic nipper；Pulp tissue is cut with ophthalmology curved scissors
Into 1mm3, it is placed in 50mL centrifuge tubes, the tissue digestion liquid of 13mL is added, it is sufficiently mixed after sealing uniformly, it is transferred to constant temperature empty
In gas shaking table, 37 DEG C, 200rpm digestion 15min；Isometric culture medium is added to blow and beat discrete cellular agglomerate repeatedly, by 70 μm
Cell screen clothes filtering, 2000rpm centrifugation 3min；Using PBS 1~2 time, precipitation is resuspended with culture medium, with 5 × 103Individual/
In mL inoculated and cultured wares, cell suspension is mixed, be put in 37 DEG C, cultivate in the CO2gas incubator that humidity is 95%.Every 24
Hour, survey a cell quantity with blood counting chamber.
4th, experimental result
With the culture medium of embodiment of the present invention 1-3 gained culture dental pulp stem cells, with comparative example 1 and the gained training of comparative example 2
The culture medium for supporting dental pulp stem cell is cultivated dental pulp stem cell, is cultivated when starting and is cultivated 1 day, 2 days, 3 days, 4 days and 5
It cell quantity is as shown in table 1.
Table 1：Different culture media is to cell quantity after dental pulp stem cell culture
As can be seen from Table 1, in identical incubation time, embodiment of the present invention 1-3 gained culture dental pulps are grown in and are done
The cell quantity of dental pulp stem cell in the culture medium of cell, hence it is evident that more than being grown in comparative example 1 and the gained culture dental pulp of comparative example 2
The cell quantity of dental pulp stem cell in the culture medium of stem cell.This explanation, the culture medium of present invention culture dental pulp stem cell can be with
The propagation of dental pulp stem cell is obviously promoted, the expanding effect of the culture medium of present invention culture dental pulp stem cell is good.
Test example two, to the dental pulp stem cell vitro differentiation after propagation be Gegenbaur's cell, chondroblast, lipoblast
Potential Analysis influence experiment
1st, tested culture medium：The culture medium of embodiment of the present invention 1-3 gained culture dental pulp stem cells, and the He of comparative example 1
The culture medium of the gained culture dental pulp stem cell of comparative example 2.
2nd, source of human stem cell is cultivated：From the dental pulp stem cell of dental pulp.
3rd, cell in vitro Analytical Chemical Experiment
Passage is expanded to P10 generations, LONZA into fat, skeletonization, into cartilage detection kit to P10 for tooth is used
Marrow stem cell carries out differentiation detection.
4th, experimental result
Testing result shows, through the dental pulp that the medium culture of embodiment of the present invention 1-3 gained culture dental pulp stem cells is crossed
Stem cells into fat cells, chondroblast, the ratio of Gegenbaur's cell is more than 85%.And comparative example 1 and the institute of comparative example 2
The dental pulp stem cell differentiation lipoblast that the medium culture of dental pulp stem cell is crossed, chondroblast, Gegenbaur's cell must be cultivated
Ratio 65%.Therefore, the culture medium of present invention gained culture dental pulp stem cell can effectively keep dental pulp stem cell
Multidirectional Differentiation ability.
Claims (8)
1. it is a kind of cultivate dental pulp stem cell culture medium, it is characterised in that including basal medium and addition it is described basis training
Support base in additive, the composition of the additive with final concentration, including：EGF 3-5 μ g/L, carboxymethyl chitosan
Sugared 2-4g/L, catalase 5-8mg/L, lipoic acid 0.14-0.18mg/L, 0.3-0.6 μM of Co-Q10, LN1 5-
25 μ g/L, 2-5 μM of leukotrienes, 8-14 μM of niacinamide, taurine 2-4mg/L, platelet derived growth factor 2-6 μ g/L, chlorination
70-80 μM of choline, diosgenin 0.5-2mg/L.
2. the culture medium of dental pulp stem cell is cultivated as claimed in claim 1, it is characterised in that including basal medium and addition
Additive in the basal medium, the composition of the additive with final concentration, including：The μ g/L of EGF 4,
Carboxymethyl chitosan 3g/L, catalase 6mg/L, lipoic acid 0.16mg/L, 0.4 μM of Co-Q10, the μ g/ of LN1 8
L, 4 μM of leukotrienes, 12 μM of niacinamide, taurine 3mg/L, the μ g/L of platelet derived growth factor 5,76 μM of Choline Chloride, Chinese yam
Sapogenin 1.4mg/L.
3. the preparation method of the culture medium of dental pulp stem cell is cultivated as claimed in claim 1 or 2, it is characterised in that step is such as
Under：
S1 in basal medium add EGF, lipoic acid, laminin, leukotrienes, niacinamide, taurine,
Platelet derived growth factor and Choline Chloride, stir 20-40 minutes, add carboxymethyl chitosan and diosgenin, continue
Stirring 30-40 minutes, stands 1-3 hours, adds catalase and Co-Q10, stirs 30 minutes, obtains mixture；
The pH to 6.8-7.1 of S2 regulating steps S1 gained mixtures, with 0.2-0.25 micron membrane filter filtration sterilizations, obtains final product.
4. the preparation method of the culture medium of dental pulp stem cell is cultivated as claimed in claim 3, it is characterised in that the step S1
Stirring 30 minutes.
5. the preparation method of the culture medium of dental pulp stem cell is cultivated as claimed in claim 3, it is characterised in that the step S1
Continue to stir 35 minutes.
6. the preparation method of the culture medium of dental pulp stem cell is cultivated as claimed in claim 3, it is characterised in that the step S1
Stand 2 hours.
7. the preparation method of the culture medium of dental pulp stem cell is cultivated as claimed in claim 3, it is characterised in that the step S2
The pH to 7.0 of regulating step S1 gained mixtures.
8. the preparation method of the culture medium of dental pulp stem cell is cultivated as claimed in claim 3, it is characterised in that the step S2
With 0.23 micron membrane filter filtration sterilization.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107828723A (en) * | 2017-11-13 | 2018-03-23 | 重庆斯德姆生物技术有限公司 | A kind of type I collagen culture medium and preparation method thereof |
| CN111955454A (en) * | 2020-05-14 | 2020-11-20 | 领航干细胞再生医学工程有限公司 | Human in-vitro tooth preservative fluid and preparation method thereof |
| CN112094804A (en) * | 2019-06-18 | 2020-12-18 | 中国医学科学院基础医学研究所 | Heterogeneous stem cell population, preparation method and application thereof |
| CN116286624A (en) * | 2023-03-14 | 2023-06-23 | 青岛安融生物健康科技有限公司 | Application of water-soluble chitosan in stem cell in-vitro culture |
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| CN101198343A (en) * | 2004-10-14 | 2008-06-11 | 生物模拟治疗公司 | Platelet-derived growth factor compositions and methods of use thereof |
| CN103060264A (en) * | 2012-12-20 | 2013-04-24 | 上海市第十人民医院 | Stem cell culture medium and application thereof and stem cell cultivation method |
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