CN112442479A - Cell culture solution and preparation method thereof - Google Patents
Cell culture solution and preparation method thereof Download PDFInfo
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- CN112442479A CN112442479A CN202011402132.9A CN202011402132A CN112442479A CN 112442479 A CN112442479 A CN 112442479A CN 202011402132 A CN202011402132 A CN 202011402132A CN 112442479 A CN112442479 A CN 112442479A
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- 238000002360 preparation method Methods 0.000 title abstract description 9
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- 229920001661 Chitosan Polymers 0.000 claims abstract description 27
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 17
- 239000011550 stock solution Substances 0.000 claims abstract description 13
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- 230000000996 additive effect Effects 0.000 claims abstract description 10
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- 239000012894 fetal calf serum Substances 0.000 claims abstract description 5
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 29
- 238000000034 method Methods 0.000 claims description 20
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- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241001506047 Tremella Species 0.000 description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 2
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- ZHJGWYRLJUCMRT-UHFFFAOYSA-N 5-[6-[(4-methylpiperazin-1-yl)methyl]benzimidazol-1-yl]-3-[1-[2-(trifluoromethyl)phenyl]ethoxy]thiophene-2-carboxamide Chemical compound C=1C=CC=C(C(F)(F)F)C=1C(C)OC(=C(S1)C(N)=O)C=C1N(C1=C2)C=NC1=CC=C2CN1CCN(C)CC1 ZHJGWYRLJUCMRT-UHFFFAOYSA-N 0.000 description 1
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- 229960003957 dexamethasone Drugs 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 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
- C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Discloses a cell culture solution, which comprises a basic culture solution and an additive, wherein the basic culture solution comprises 85-95 v% of DMEM culture solution, 5-15 v% of fetal calf serum and 5-15mL/L of double-antibody stock solution; the additive is selected from chitosan modified nano-hydroxyapatite. In addition, a preparation method thereof is also disclosed. Aiming at osteoblasts, the cell culture solution has the effect of promoting cell proliferation and has a more remarkable effect of promoting cell mineralization.
Description
Technical Field
The invention belongs to the technical field of animal cell culture; relates to a cell culture solution and a preparation method thereof.
Background
Cell culture refers to the growth of cells under in vitro conditions. There are three survival conditions for in vitro cultured cells. The first is nutrition. The nutrients required for in vitro cell culture mainly include sugars, amino acids and vitamins. Synthetic media are most often used to provide the above nutrients, but human or animal serum, plasma, fetal juices, etc. are still added. For example, to allow cells to grow normally, 10-15% calf serum is typically added. The second is the environment. Sterility is the primary condition for ensuring the survival of cultured cells. The culture broth is a high nutrient not only for cells but also for bacteria and molds. The partially contaminating microorganisms multiply even faster than the cells and produce toxins that cause cell death. The optimum temperature for cell culture is 35-37 deg.C; the gas required is mainly O2And CO2(ii) a The pH value is 7.0-7.4. Whether the culture vessel is treated or not has great influence on the adherent growth of the cells. With the exception of a few cells grown in suspension, the vast majority of cells belong to anchorage-dependent cells or cultures. They can grow, survive or otherwise survive only when attached to the surface of an item that is not chemically activeThe function is maintained.
Osteoblasts are the primary functional cells for bone formation and are responsible for the synthesis, secretion and mineralization of bone matrix. The osteoblasts are developed from multipotent mesenchymal stem cells under the regulation of various regulatory factors in vivo, and the regulatory factors mainly comprise BMP-2; which can induce differentiation of stromal cells into osteoblasts. Osteoblasts are spindle, cone or cube shaped, cytoplasmic basophilic. The nucleus is located at one end of the cell, the nucleolus is obvious, and the surface has short protrusions connected with adjacent cells.
Mills et al, 1979, first reported the successful culture of human osteoblasts by in vitro tissue mass culture; subsequently, others reported successful osteoblast culture. The main culture method comprises an enzyme digestion method, a tissue block method and an improved enzyme digestion-tissue block method combined culture method.
The conditions required for culturing osteoblasts are the same as those required for culturing ordinary cells, and the nutrition and environmental conditions are required to meet the requirements of cell culture.
Chinese patent application CN105567629A discloses a culture medium for culturing osteoblasts in vitro. The method is characterized in that: comprises 10.0-15.0g/L of basic culture medium, 300mg/L of tremella polysaccharide, 8-10mL of serum, a proper amount of sodium bicarbonate for adjusting the pH value to 7.2-7.6, and water for fixing the volume to 1L; the basic culture medium is DMEM, alpha-MEM, RPMI-1640, F12 and DMEM/F12; the purity of the tremella polysaccharide is more than 75%; the serum is fetal calf serum, human serum, horse serum and sheep serum; the culture medium is used for culturing osteoblasts in vitro, can accelerate the growth of the osteoblasts and can reduce the use amount of serum.
Sunxikun research shows that the nano hydroxyapatite ceramic is leached by using alpha-MEM containing 10% FBS as a complete culture medium for 72h to obtain a leaching solution, the proliferation rate of the obtained osteoblasts is 98.4%, and the obtained osteoblasts have no obvious difference compared with the blank group of 100%. In addition, the nano hydroxyapatite ceramic supports the attachment and growth of osteoblasts and has a good effect of promoting cell mineralization, but the effect is not significant enough.
Aiming at the defects in the prior art, a cell culture solution with more obvious osteoblast culture effect and a preparation method thereof are urgently needed to be found.
Disclosure of Invention
An object of the present invention is to provide a cell culture solution. Aiming at osteoblast precursor cells, the cell culture solution has a remarkable effect of promoting cell proliferation and a more remarkable effect of promoting cell mineralization.
Another object of the present invention is to provide a method for preparing the above-mentioned cell culture solution. The preparation process is simple and easy to operate, and is suitable for large-scale production.
In order to achieve the above objects, in one aspect, the present invention provides a cell culture solution, comprising a basic culture solution and additives, wherein the basic culture solution comprises 85-95 v% DMEM culture solution, 5-15 v% fetal bovine serum and 5-15mL/L double antibody stock solution; the method is characterized in that the additive is selected from chitosan modified nano hydroxyapatite.
Preferably, the basic culture solution comprises 88-92 v% DMEM culture solution, 8-12 v% fetal bovine serum and 8-12mL/L double antibody stock solution.
In a specific embodiment, the basal medium comprises 90 v% DMEM medium and 10 v% fetal bovine serum and 10mL/L double antibody stock solution.
The cell culture solution according to the present invention, wherein the chitosan is selected from the group consisting of chitosan having a self-adhesive average molecular weight Mv of 2-3.5 × 105Dalton, degree of deacetylation DD 65-85%.
Preferably, the chitosan is selected from the group consisting of chitosan having a self-average molecular weight Mv of 2.5-3 × 105Dalton, degree of deacetylation DD 70-80%.
In a particular embodiment, the chitosan is selected from the group consisting of chitosan having a self-average molecular weight Mv of 2.72 × 105Dalton, degree of deacetylation DD 74.7%.
The cell culture solution is characterized in that the nano hydroxyapatite is selected from carbonate substituted B-type hydroxyapatite.
In the present invention, the carbonate-substituted hydroxyapatite includes type A hydroxyapatite and type C hydroxyapatite in addition to type B hydroxyapatite. The hydroxyapatite is classified according to the kind of anion of the carbonate substituted hydroxyapatite. The A type hydroxyapatite represents that carbonate is only substituted for OH of hydroxyapatite-(ii) a Hydroxyapatite type B represents PO in which carbonate is substituted only for hydroxyapatite4 3-(ii) a The C-type hydroxyapatite represents that carbonate replaces OH of the hydroxyapatite at the same time-And PO4 3-。
The cell culture solution provided by the invention has the carbonate content of the B-type hydroxyapatite of 8-18 wt%.
Preferably, the carbonate content of the hydroxyapatite type B is between 10 and 15 wt%.
In a particular embodiment, the carbonate content of hydroxyapatite type B is 12.8 wt%.
The cell culture solution provided by the invention is characterized in that the weight ratio of chitosan to nano-hydroxyapatite is 1: (10-30).
Preferably, the weight ratio of the chitosan to the nano hydroxyapatite is 1: (15-25).
In a specific embodiment, the weight ratio of chitosan to nano-hydroxyapatite is 1: 20.
the cell culture solution of the present invention, wherein the additive is added in an amount of 5 to 50mg/L based on the volume of the basic culture solution.
Preferably, the additive is added in an amount of 10-40mg/L based on the volume of the basic culture solution.
In a specific embodiment, the additive is added in an amount of 20mg/L based on the volume of the basic culture solution.
The cell culture solution of the present invention is prepared by the following method: 13.4g of high-sugar DMEM dry powder is dissolved in 950ml of triple-distilled water, 3.7g of sodium bicarbonate is added, N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES) buffer is added to adjust the pH value to 7.2, and the volume is adjusted to 1L.
The cell culture solution of the invention, wherein the preparation method of the double antibody stock solution is as follows: 80 ten thousand units of penicillin and 0.8g streptomycin were dissolved in 80mL PBS buffer, filtered through a microporous membrane, and stored at-20 ℃.
On the other hand, the invention also provides a preparation method of the cell culture solution, which comprises the following steps:
preparing DMEM culture solution and double-antibody stock solution;
mixing the DMEM culture solution and fetal calf serum according to a formula, and adding the double-antibody stock solution according to a proportion to obtain a basic culture solution;
and adding the prepared chitosan modified nano-hydroxyapatite into the basic culture solution, and stirring to uniformly disperse the chitosan modified nano-hydroxyapatite to obtain the cell culture solution.
The invention has the beneficial effects that:
(1) aiming at osteoblasts, the cell culture solution has a remarkable effect of promoting cell proliferation and a more remarkable effect of promoting cell mineralization.
(2) The preparation method of the cell culture solution is simple and easy to operate, and is suitable for large-scale production.
Detailed Description
The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the invention.
Example 1
(1) Preparing nano hydroxyapatite: 11.8g (50mmol) of calcium nitrate tetrahydrate (analytical grade) was added to 80mL of acetone (analytical grade), and the mixture was dissolved with stirring to obtain a calcium nitrate solution. To 200mL of distilled water, 3.96g (30mmol) of diammonium hydrogen phosphate (analytically pure) and 1.19g (15mmol) of ammonium hydrogen carbonate (analytically pure) were added, and the mixture was dissolved by stirring, and the pH was adjusted to 11.0. And mixing the two solutions under the stirring condition, and stirring for 30s after the mixing is finished to obtain the nano-hydroxyapatite suspension. The average particle size of the nano-hydroxyapatite measured by a dynamic light scattering DLS method is 20 nm. Filtration was performed immediately using Millipore all-glass membrane-change filters and washed three times with triple distilled water. And finally, freeze-drying the nano-hydroxyapatite suspension to obtain the nano-hydroxyapatite. The carbonate substitution pattern was confirmed to be hydroxyapatite type B using a literature (J Mater Sci: Mater Med 29,103) method and the carbonate content was calculated to be 12.8 wt%.
(2) Preparing chitosan modified nano hydroxyapatite: and adding 300mg of carbonate substituted B-type nano-hydroxyapatite into 12mL of triple distilled water, and performing ultrasonic treatment to uniformly disperse the carbonate substituted B-type nano-hydroxyapatite to obtain a dispersion liquid. Another 15mg of chitosan (viscosity average molecular weight Mv 2.72X 10)5Dalton, degree of deacetylation DD ═ 74.7%) was dissolved in 12mL of a 1% aqueous solution of acetic acid, to give a chitosan solution. And then adding the chitosan solution into the dispersion liquid, ultrasonically dispersing for 60min, standing for 2h, and adjusting the pH value to 7.0 to obtain the chitosan modified nano-hydroxyapatite dispersion liquid. And (5) freeze-drying to obtain the chitosan modified nano-hydroxyapatite.
(3) Preparing a DMEM culture solution: 13.4g of high-sugar DMEM dry powder is dissolved in 950ml of triple-distilled water, 3.7g of sodium bicarbonate is added, N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES) buffer is added to adjust the pH value to 7.2, and the volume is adjusted to 1L.
(4) Preparing a double-antibody stock solution: 80 ten thousand units of penicillin and 0.8g streptomycin were dissolved in 80mL PBS buffer, filtered through a microporous membrane, and stored at-20 ℃.
(5) Preparing a basic culture solution: 90 parts by volume of DMEM culture solution and 10 parts by volume of fetal calf serum are mixed, and 10mL/L of double antibody stock solution is added to the mixture to obtain basic culture solution.
(6) Adding chitosan modified nano hydroxyapatite into the basic culture solution, wherein the addition amount of the additive is 20mg/L based on the volume of the basic culture solution; the mixture was uniformly dispersed by stirring to obtain the cell culture solution of example 1.
Comparative example 1
Based on the step (1) of the method in the embodiment 1, the ammonium bicarbonate is omitted, and the non-carbonate substituted nano-hydroxyapatite is obtained. The average particle size of the nano hydroxyapatite measured by a dynamic light scattering DLS method is 24 nm.
The procedure was the same as in example 1, and the cell culture solution of comparative example 1 was finally obtained.
Comparative example 2
On the basis of the method of example 1, step (2) is omitted, i.e., no chitosan modification is performed; and directly adding carbonate substituted B-type nano hydroxyapatite in the step (6).
The procedure was the same as in example 1, and the cell culture solution of comparative example 2 was finally obtained.
Cell proliferation Properties
The cell proliferation performance adopts a tetrazolium salt (MTT) colorimetric method, and the specific method comprises the following steps: logarithmic growth of mouse embryonic osteoblast precursor cells (MC3T3-E1, available from Shanghai Pont industries, Ltd.) at 5X 103Per cm2Seeded in 96-well plates. After 24 hours of cell attachment, the cell culture solutions of example 1, comparative example 1 and comparative example 2 were added, respectively; the cell culture solution of example 1 without cells was used as a control group. Each test group or control group was provided with 10 wells. After 5 days of culture, the cell culture medium was aspirated. PBS buffer was washed 3 times. mu.L of MTT reagent was added to each well and incubated at 37 ℃ for 4 h. Then 80. mu.L DMSO was added and shaken gently for 10min to mix the solution completely and uniformly. And measuring the light absorption value of the microplate reader at the wavelength of 490 nm. The relative cell proliferation rate R (%) of example 1 and comparative examples 1-2 was calculated as R ═ ODTest group/ODControl groupX 100%, the results are averaged.
Cell mineralization Properties
Logarithmic growth of mouse embryonic osteoblast precursor cells (MC3T3-E1) at 1X 104Per cm2Seeded in 24-well plates. Cells were switched to osteogenic induction medium 24 hours after adherence. The osteogenesis inducing culture solution was divided into 4 groups, and 50mg/L of vitamin C, 10mmol/L of P-phosphoglycerol and 1X 10 of the amount of glycerol were added to the cell culture solutions of example 1, comparative example 1 and comparative example 2 and the basal culture solution of example 1 (control group), respectively-7Obtaining mol/L dexamethasone; each group had 6 wells, and the solution was changed for 3 days. After 28 days of culture, calcium nodules were quantitatively identified by ALP and von kossa double-staining co-localization method, and the results were averaged to obtain the number of calcium nodules per well.
The above results are shown in table 1 below.
TABLE 1
| Relative increment rate R (%) | Calcium nodule (single/hole) | |
| Example 1 | 143 | 31 |
| Comparative example 1 | 124 | 22.5 |
| Comparative example 2 | 118 | 18.67 |
| Control group | 100 | 7.83 |
As can be seen from table 1, the cell culture fluid of example 1 of the present invention has a more significant effect of promoting cell proliferation and a more significant effect of promoting cell mineralization with respect to osteoblast precursor cells than comparative examples 1 and 2.
It should be understood that the detailed description of the invention is merely illustrative of the spirit and principles of the invention and is not intended to limit the scope of the invention. Furthermore, it should be understood that various changes, substitutions, deletions, modifications or adjustments may be made by those skilled in the art after reading the disclosure of the present invention, and such equivalents are also within the scope of the invention as defined in the appended claims.
Claims (10)
1. A cell culture solution comprises a basic culture solution and an additive, wherein the basic culture solution comprises 85-95 v% of DMEM culture solution, 5-15 v% of fetal bovine serum and 5-15mL/L of double-antibody stock solution; the method is characterized in that the additive is selected from chitosan modified nano hydroxyapatite.
2. The cell culture solution according to claim 1, wherein the chitosan is selected from the group consisting of chitosan having an average molecular weight of Mv 2-3.5 x 105Dalton, degree of deacetylation DD 65-85%.
3. The cell culture fluid of claim 1, wherein the nano-hydroxyapatite is selected from carbonate substituted hydroxyapatite type B.
4. The cell culture solution according to claim 3, wherein the carbonate content of the hydroxyapatite type B is 8 to 18 wt%.
5. The cell culture solution according to claim 4, wherein the carbonate content of the hydroxyapatite type B is 12.8 wt%.
6. The cell culture solution of claim 1, wherein the weight ratio of the chitosan to the nano-hydroxyapatite is 1: (10-30).
7. The cell culture solution according to claim 1, wherein the additive is added in an amount of 5 to 50mg/L based on the volume of the basic culture solution.
8. The cell culture solution according to claim 1, wherein the DMEM solution is prepared by the following method: 13.4g of high-sugar DMEM dry powder is dissolved in 950ml of triple-distilled water, 3.7g of sodium bicarbonate is added, N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid (HEPES) buffer is added to adjust the pH value to 7.2, and the volume is adjusted to 1L.
9. The cell culture solution according to claim 1, wherein the double antibody stock solution is prepared by the following method: 80 ten thousand units of penicillin and 0.8g streptomycin were dissolved in 80mL PBS buffer, filtered through a microporous membrane, and stored at-20 ℃.
10. A method for preparing a cell culture fluid according to any one of claims 1 to 9, comprising the steps of:
preparing DMEM culture solution and double-antibody stock solution;
mixing the DMEM culture solution and fetal calf serum according to a formula, and adding the double-antibody stock solution according to a proportion to obtain a basic culture solution;
and adding the prepared chitosan modified nano-hydroxyapatite into the basic culture solution, and stirring to uniformly disperse the chitosan modified nano-hydroxyapatite to obtain the cell culture solution.
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| US20140178455A1 (en) * | 2012-12-21 | 2014-06-26 | University Of Connecticut | Gradient Porous Scaffolds |
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| US20140178455A1 (en) * | 2012-12-21 | 2014-06-26 | University Of Connecticut | Gradient Porous Scaffolds |
| CN107854732A (en) * | 2017-11-01 | 2018-03-30 | 哈尔滨市第医院 | Improve space and hole promotes the compound rest and preparation method of cell adhesion rate |
| US20210162093A1 (en) * | 2018-09-29 | 2021-06-03 | Guangzhou Beogene Biotechnology Co., Ltd. | Absorbable bone wax having function of promoting bone repair and preparation method thereof |
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