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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- stem cell
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- 210000005258 dental pulp stem cell Anatomy 0.000 title claims abstract description 67
- 239000001963 growth medium Substances 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000007640 basal medium Substances 0.000 claims abstract description 32
- 239000000203 mixture Substances 0.000 claims abstract description 25
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229920001661 Chitosan Polymers 0.000 claims abstract description 18
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims abstract description 17
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 claims abstract description 17
- DWCSNWXARWMZTG-UHFFFAOYSA-N Trigonegenin A Natural products CC1C(C2(CCC3C4(C)CCC(O)C=C4CCC3C2C2)C)C2OC11CCC(C)CO1 DWCSNWXARWMZTG-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000000654 additive Substances 0.000 claims abstract description 16
- 230000000996 additive effect Effects 0.000 claims abstract description 16
- WQLVFSAGQJTQCK-VKROHFNGSA-N diosgenin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CC[C@H](O)CC4=CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 WQLVFSAGQJTQCK-VKROHFNGSA-N 0.000 claims abstract description 16
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims abstract description 15
- 235000019743 Choline chloride Nutrition 0.000 claims abstract description 15
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims abstract description 15
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 claims abstract description 15
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 claims abstract description 15
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims abstract description 15
- 229960003178 choline chloride Drugs 0.000 claims abstract description 15
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 claims abstract description 15
- 150000002617 leukotrienes Chemical class 0.000 claims abstract description 15
- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 claims abstract description 15
- 235000019136 lipoic acid Nutrition 0.000 claims abstract description 15
- 229960003966 nicotinamide Drugs 0.000 claims abstract description 15
- 235000005152 nicotinamide Nutrition 0.000 claims abstract description 15
- 239000011570 nicotinamide Substances 0.000 claims abstract description 15
- 229960002663 thioctic acid Drugs 0.000 claims abstract description 15
- 229960003080 taurine Drugs 0.000 claims abstract description 11
- 102000016938 Catalase Human genes 0.000 claims abstract description 10
- 108010053835 Catalase Proteins 0.000 claims abstract description 10
- 238000003756 stirring Methods 0.000 claims description 18
- 238000001914 filtration Methods 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 7
- 230000001105 regulatory effect Effects 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- 239000012467 final product Substances 0.000 claims description 5
- 239000006143 cell culture medium Substances 0.000 claims description 3
- 238000012549 training Methods 0.000 claims description 2
- 235000002722 Dioscorea batatas Nutrition 0.000 claims 1
- 235000006536 Dioscorea esculenta Nutrition 0.000 claims 1
- 240000001811 Dioscorea oppositifolia Species 0.000 claims 1
- 235000003416 Dioscorea oppositifolia Nutrition 0.000 claims 1
- 238000005660 chlorination reaction Methods 0.000 claims 1
- 229960001231 choline Drugs 0.000 claims 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 claims 1
- NWMIYTWHUDFRPL-UHFFFAOYSA-N sapogenin Natural products COC(=O)C1(CO)C(O)CCC2(C)C1CCC3(C)C2CC=C4C5C(C)(O)C(C)CCC5(CCC34C)C(=O)O NWMIYTWHUDFRPL-UHFFFAOYSA-N 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 19
- 230000003321 amplification Effects 0.000 abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 6
- 238000004113 cell culture Methods 0.000 abstract description 5
- 230000000052 comparative effect Effects 0.000 description 12
- 210000003074 dental pulp Anatomy 0.000 description 8
- 230000004069 differentiation Effects 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 239000001257 hydrogen Substances 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 150000002978 peroxides Chemical class 0.000 description 5
- 210000000130 stem cell Anatomy 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 4
- 239000005864 Sulphur Substances 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 210000001612 chondrocyte Anatomy 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
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- 238000011069 regeneration method Methods 0.000 description 3
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- IXQKXEUSCPEQRD-NRNCYQGDSA-N (2S,4R,23E)-2,16beta,20-trihydroxy-9beta,10,14-trimethyl-1,11,22-trioxo-4,9-cyclo-9,10-secocholesta-5,23-dien-25-yl acetate Chemical compound C([C@H]1[C@]2(C)C[C@@H](O)[C@@H]([C@]2(CC(=O)[C@]11C)C)[C@@](C)(O)C(=O)C=CC(C)(C)OC(=O)C)C=C2[C@H]1C[C@H](O)C(=O)C2(C)C IXQKXEUSCPEQRD-NRNCYQGDSA-N 0.000 description 2
- IHTCCHVMPGDDSL-ZJNDIJRCSA-N Cucurbitacin A Natural products O=C([C@@](O)(C)[C@H]1[C@@H](O)C[C@]2(C)[C@]1(C)CC(=O)[C@]1(CO)[C@H]2CC=C2C(C)(C)C(=O)[C@H](O)C[C@@H]12)/C=C/C(OC(=O)C)(C)C IHTCCHVMPGDDSL-ZJNDIJRCSA-N 0.000 description 2
- CVKKIVYBGGDJCR-SXDZHWHFSA-N Cucurbitacin B Natural products CC(=O)OC(C)(C)C=CC(=O)[C@@](C)(O)[C@@H]1[C@@H](O)C[C@]2(C)C3=CC[C@@H]4C(C)(C)C(=O)[C@H](O)C[C@@]4(C)[C@@H]3CC(=O)[C@@]12C CVKKIVYBGGDJCR-SXDZHWHFSA-N 0.000 description 2
- QZJJDOYZVRUEDY-UHFFFAOYSA-N Dihydrocucurbitacin B Natural products CC12C(=O)CC3(C)C(C(C)(O)C(=O)CCC(C)(C)OC(=O)C)C(O)CC3(C)C1CC=C1C2CC(O)C(=O)C1(C)C QZJJDOYZVRUEDY-UHFFFAOYSA-N 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 210000002709 dental papilla cell Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
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- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
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- 210000003716 mesoderm Anatomy 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 206010063413 odontogenic cyst Diseases 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- FUFLCEKSBBHCMO-UHFFFAOYSA-N 11-dehydrocorticosterone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 FUFLCEKSBBHCMO-UHFFFAOYSA-N 0.000 description 1
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical group C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 1
- MFYSYFVPBJMHGN-ZPOLXVRWSA-N Cortisone Chemical compound O=C1CC[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 MFYSYFVPBJMHGN-ZPOLXVRWSA-N 0.000 description 1
- MFYSYFVPBJMHGN-UHFFFAOYSA-N Cortisone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 MFYSYFVPBJMHGN-UHFFFAOYSA-N 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 229960004544 cortisone Drugs 0.000 description 1
- 239000005548 dental material Substances 0.000 description 1
- 210000001968 dental pulp cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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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
- C12N5/0662—Stem cells
- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
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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 | A kind of heterogeneous stem cell population, its preparation method and use |
| 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 |
| CN104711219A (en) * | 2015-03-01 | 2015-06-17 | 安徽新生命干细胞科技有限公司 | Dental pulp stem cell culture medium |
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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 |
| CN104711219A (en) * | 2015-03-01 | 2015-06-17 | 安徽新生命干细胞科技有限公司 | Dental pulp stem cell culture medium |
Cited By (6)
| 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 |
| CN112094804A (en) * | 2019-06-18 | 2020-12-18 | 中国医学科学院基础医学研究所 | A kind of heterogeneous stem cell population, its preparation method and use |
| CN112094804B (en) * | 2019-06-18 | 2024-05-14 | 中国医学科学院基础医学研究所 | Heterogeneous stem cell population, preparation method and application 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 |
| 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 |
| CN116286624B (en) * | 2023-03-14 | 2023-11-10 | 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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