CN111411070A - Substrate material of culture medium, preparation method thereof, culture medium and application thereof - Google Patents
Substrate material of culture medium, preparation method thereof, culture medium and application thereof Download PDFInfo
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- CN111411070A CN111411070A CN202010414203.0A CN202010414203A CN111411070A CN 111411070 A CN111411070 A CN 111411070A CN 202010414203 A CN202010414203 A CN 202010414203A CN 111411070 A CN111411070 A CN 111411070A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Abstract
The invention is suitable for the technical field of cell culture, and provides a basic substance of a culture medium, a preparation method thereof, the culture medium and application thereof, wherein the preparation method of the basic substance comprises the following steps: after the oxygen carrier is subjected to disinfection and sterilization and oxygen exposure treatment, adding a hydrogel solution for covering treatment to obtain the base substance; the oxygen carrier is one or more of perfluorocarbon compounds, artificial red blood cells and modified hemoglobin solution. The substrate substance provided by the embodiment of the invention contains the oxygen carrier and the hydrogel which is covered with the oxygen carrier, and can be added into the culture medium for continuous oxygen supply, so that the culture efficiency of the culture medium on cells such as animals and the like can be greatly improved.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a basic substance of a culture medium, a preparation method of the basic substance, the culture medium and application of the culture medium.
Background
Perfluorotributylamine (PFTBA) is a non-toxic, highly oxygen-soluble perfluorinated compound that is approximately 25 times more soluble in oxygen than water and 3 times more soluble in blood. Based on the advantages, PFTBA is widely applied to improve the early oxygen supply problem of various tissue engineering scaffolds, is used for solving the problem of oxygen supply between tissues in vivo in the recovery period after bone injury, and achieves good effect. However, PFTBA does not solve the general problem of culturing cells in vitro, and it is difficult to control oxygen concentration.
In addition, the prior art discloses an oxygen carrier for in vitro cell culture, which comprises components such as fluorocarbon, ethanol, liquid alkane, cyclic ether and the like, and although the oxygen carrier can be used for in vitro cell culture, the oxygen carrier adopts ethanol as a solvent, so that the oxygen carrier can cause damage to the cell culture, and the oxygen carrier for in vitro cell culture has the problems of low culture efficiency and the like.
Disclosure of Invention
The embodiment of the invention aims to provide a preparation method of a substrate material of a culture medium, aiming at solving the problems in the background art.
The embodiment of the invention is realized in such a way that the preparation method of the substrate substance of the culture medium comprises the following steps:
after the oxygen carrier is subjected to disinfection and sterilization and oxygen exposure treatment, adding a hydrogel solution for covering treatment to obtain the base substance; the oxygen carrier is one or more of perfluorocarbon compounds, artificial red blood cells and modified hemoglobin solution.
As a preferred embodiment of the present invention, the hydrogel solution includes all materials capable of forming hydrogel, specifically, the hydrogel includes but is not limited to one or more of agarose, collagen, alginate, hyaluronic acid, methacrylate gelatin, polyethylene glycol and cellulose.
As another preferable scheme of the embodiment of the present invention, the mass concentration of the hydrogel in the hydrogel solution is 2% to 5%.
As another preferred version of this embodiment of the present invention, the perfluorocarbon compound includes, but is not limited to, one or more of perfluorodecahydronaphthalene, perfluorotributylamine, perfluorotripropylamine, perfluoro-N- (4-methylcyclohexyl) piperidine, and perfluorobromoalkane.
As another preferable mode of the embodiment of the present invention, the modified hemoglobin solution is an organic matter cross-linked modified hemoglobin solution; the organic substance includes, but is not limited to, one of raffinose, polyethylene glycol, disuccinimidyl suberate, and glutaraldehyde.
In another preferable embodiment of the present invention, in the step, the gas subjected to the oxygen exposure treatment is a mixed gas of oxygen and carbon dioxide, and the pressure of the oxygen exposure treatment is 0.08 to 0.12 MPa.
As another preferable scheme of the embodiment of the invention, the volume ratio of the oxygen to the carbon dioxide is (93-97) to (3-7).
Another object of an embodiment of the present invention is to provide a substrate material obtained by the above-mentioned preparation method.
Another object of an embodiment of the present invention is a culture medium comprising the above-described substrate material.
Another object of an embodiment of the present invention is to provide a use of the above medium for animal cell culture.
The substrate material of the culture medium provided by the embodiment of the invention contains the oxygen carrier and is covered with the hydrogel, and the hydrogel can be added into the culture medium for continuous oxygen supply, so that the culture efficiency of the culture medium on cells such as animals and the like can be greatly improved.
Drawings
FIG. 1 is a graph showing the comparison of cell viability between the oxygen carrier-cultured group and the control group.
FIG. 2 is a graph comparing the soft agarose cloning experiments of the oxygen carrier culture group and the control group.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
This embodiment provides a method for preparing a substrate material for a culture medium, comprising the steps of:
firstly, adding oxygen carriers into the bottom wall and the side wall of the culture hole, sterilizing the oxygen carriers by using ultraviolet rays, and then introducing mixed gas of oxygen and carbon dioxide (the volume ratio of the oxygen to the carbon dioxide is 93: 7) with the air pressure of 0.08MPa for oxygen exposure treatment; then, a hydrogel solution with a mass concentration of 2% is added to the culture well to perform covering treatment, and the substrate substance can be obtained. The substrate substance is added into a basic culture solution to prepare a culture medium for in vitro culture of animal cells, the mass concentration of oxygen carriers in the culture medium is controlled to be 1%, and the basic culture solution can adopt the existing commercially available MEM culture medium.
Wherein the oxygen carrier is a modified hemoglobin solution; the hydrogel in the hydrogel solution is agarose; the preparation method of the hydrogel solution comprises the following steps: selecting liquid with the same components as the basic culture solution as a solvent, adding 2% of hydrogel, and heating and stirring to obtain the hydrogel solution.
In addition, the modified hemoglobin solution is a hemoglobin solution modified by organic matter crosslinking; the organic matter is raffinose. The modification of modified hemoglobin solutions is prior art and comprises the steps of: adding distilled water into expired red blood cells or animal red blood cells, swelling, removing cell membranes, and crosslinking subunits of hemoglobin by using raffinose as an intramolecular crosslinking agent to enhance the stability of hemoglobin, thereby obtaining a modified hemoglobin solution.
Example 2
This embodiment provides a method for preparing a substrate material for a culture medium, comprising the steps of:
firstly, adding an oxygen carrier into the bottom wall and/or the side wall of a culture bottle, sterilizing the oxygen carrier by using a steam high-temperature sterilization method, and then introducing mixed gas of oxygen and carbon dioxide (the volume ratio of the oxygen to the carbon dioxide is 97: 3) with the air pressure of 0.12MPa for oxygen exposure treatment; then, a hydrogel solution with a mass concentration of 5% is added to the culture flask for covering treatment, and the substrate substance can be obtained. The substrate substance can be added into basic culture solution to obtain culture medium for in vitro culture of animal cells, wherein the mass concentration of oxygen carrier in the culture medium is controlled at 20%, and the basic culture solution can be F12 culture medium available in the market.
Wherein the oxygen carrier is a mixture of artificial red blood cells and a modified hemoglobin solution. The hydrogel in the hydrogel solution is a mixture of collagen and alginate, and the preparation method of the hydrogel solution comprises the following steps: selecting liquid with the same components as the basic culture solution as a solvent, adding 5% of hydrogel, and heating and stirring to obtain the hydrogel solution.
In addition, the artificial red blood cells are commercially available products. The modified hemoglobin solution is an organic matter cross-linking modified hemoglobin solution; the organic substance is polyethylene glycol. The modification of modified hemoglobin solutions is prior art and comprises the steps of: adding distilled water into overdue red blood cells or animal red blood cells, swelling, removing cell membranes, and crosslinking subunits of hemoglobin by using polyethylene glycol as an intramolecular crosslinking agent to enhance the stability of hemoglobin to obtain a modified hemoglobin solution.
Example 3
This embodiment provides a method for preparing a substrate material for a culture medium, comprising the steps of:
firstly, adding an oxygen carrier into the bottom wall of a culture hole formed by sintering Teflon, sterilizing the oxygen carrier by using ultraviolet rays, and then introducing mixed gas of oxygen and carbon dioxide (the volume ratio of the oxygen to the carbon dioxide is 95: 5) with the air pressure of 0.1MPa for oxygen exposure treatment; then, adding a hydrogel solution with the mass concentration of 3% into the culture holes for covering treatment, and obtaining the substrate substance. The basic substance is added into basic culture solution to obtain culture medium for in vitro culture of animal cells, the mass concentration of oxygen carrier in the culture medium is controlled at 10%, and the basic culture solution can be DMEM culture medium available in the market.
Wherein the oxygen carrier is perfluorocarbon compound, and the perfluorocarbon compound is perfluorodecalin. The hydrogel in the hydrogel solution is a mixture of hyaluronic acid, methacrylate gelatin, polyethylene glycol and cellulose, and the preparation method of the hydrogel solution comprises the following steps: selecting liquid with the same components as the basic culture solution as a solvent, adding 3% of hydrogel, and heating and stirring to obtain the hydrogel solution.
In addition, the culture hole is formed by sintering Teflon, so that the oxygen carrier and the bottom wall of the culture hole can be infiltrated, and the influence of surface tension on the hydrogel layer to be formed on the upper layer can be avoided.
Example 4
This embodiment provides a method for preparing a substrate material for a culture medium, comprising the steps of:
firstly, adding an oxygen carrier into the side wall of a culture hole formed by sintering Teflon, sterilizing the oxygen carrier by using ultraviolet rays, introducing mixed gas of oxygen and carbon dioxide (the volume ratio of the oxygen to the carbon dioxide is 95: 5) with the air pressure of 0.1MPa for aeration treatment, then adding hydrogel solution with the mass concentration of 3% into the culture hole for covering treatment to obtain a substrate substance, adding the substrate substance into basic culture solution to prepare a culture medium for in-vitro culture of animal cells, wherein the mass concentration of the oxygen carrier in the culture medium is controlled at 10%, and the basic culture solution can adopt the existing commercially available L15 culture medium.
Wherein the oxygen carrier is a mixture of perfluorocarbon compounds, artificial erythrocytes and modified hemoglobin solution, and the perfluorocarbon compounds are a mixture of perfluorotributylamine and perfluorotripropylamine. The hydrogel in the hydrogel solution is a mixture of polyethylene glycol and cellulose, and the preparation method of the hydrogel solution comprises the following steps: selecting liquid with the same components as the basic culture solution as a solvent, adding 3% of hydrogel, and heating and stirring to obtain the hydrogel solution.
In addition, the artificial red blood cells are commercially available products. The modified hemoglobin solution is an organic matter cross-linking modified hemoglobin solution; the organic substance is disuccinimidyl suberate. The modification of modified hemoglobin solutions is prior art and comprises the steps of: adding distilled water into expired red blood cells or animal red blood cells, swelling, removing cell membranes, and crosslinking subunits of hemoglobin by using disuccinimidyl suberate as an intramolecular crosslinking agent to enhance the stability of hemoglobin to obtain a modified hemoglobin solution.
Example 5
This embodiment provides a method for preparing a substrate material for a culture medium, comprising the steps of:
firstly, adding an oxygen carrier into the side wall of a culture hole formed by sintering Teflon, sterilizing the oxygen carrier by using ultraviolet rays, introducing mixed gas of oxygen and carbon dioxide (the volume ratio of the oxygen to the carbon dioxide is 95: 5) with the air pressure of 0.1MPa for aeration treatment, then adding hydrogel solution with the mass concentration of 3% into the culture hole for covering treatment to obtain a substrate substance, adding the substrate substance into basic culture solution to prepare a culture medium for in-vitro culture of animal cells, wherein the mass concentration of the oxygen carrier in the culture medium is controlled at 10%, and the basic culture solution can adopt the existing commercially available L15 culture medium.
Wherein the oxygen carrier is one or more of perfluorocarbon compounds, artificial erythrocytes and modified hemoglobin solution, and the perfluorocarbon compounds are a mixture of perfluorodecalin, perfluoro-N- (4-methylcyclohexyl) piperidine and perfluorobromoalkane. The hydrogel in the hydrogel solution is collagen, and the preparation method of the hydrogel solution comprises the following steps: selecting liquid with the same components as the basic culture solution as a solvent, adding 3% of hydrogel, and heating and stirring to obtain the hydrogel solution.
In addition, the artificial red blood cells are commercially available products. The modified hemoglobin solution is an organic matter cross-linking modified hemoglobin solution; the organic substance is glutaraldehyde. The modification of modified hemoglobin solutions is prior art and comprises the steps of: adding distilled water into expired red blood cells or animal red blood cells, swelling, removing cell membranes, and crosslinking subunits of hemoglobin by using glutaraldehyde as an intramolecular crosslinking agent to enhance the stability of hemoglobin, thereby obtaining a modified hemoglobin solution.
The animal cells are placed in the culture medium prepared in the embodiment 5 for culture, and Superoxide dismutase (SOD) can be added in the culture process to prevent the damage of Superoxide to the cells caused by overhigh oxygen partial pressure, wherein the oxygen release period of the culture medium reaches 4-6 days, the limitation of closed culture for 3 days is exceeded, the liquid amount of the culture can reach 150-300% of the original liquid amount, the culture time can be prolonged to 130-200% of the original liquid amount, and the cell amount harvested in one-time culture is 180-400% of the original cell amount, so that the efficiency of in-vitro culture of the animal cells can be greatly improved.
In addition, L ewis (lung cancer cells) cultured in a conventional medium containing a basic culture solution is used as a control group, L ewis (lung cancer cells) cultured in the medium provided in the above example 5 is used as an oxygen carrier culture group, the cell viability after the culture is detected by using the MTT method, specifically, 6 × 103L ewis (lung cancer cells) cells are inoculated into a 96-well plate containing the medium, the culture is cultured for 24h, MTT reagent is added for incubation for 4h, the supernatant is removed, dimethyl sulfoxide is added, a micro-oscillator is shaken to dissolve crystals, an microplate reader is used for detecting the OD570nm value, and the measured OD value is the cell viability, wherein the comparison result of the cell viability experiments of the oxygen carrier culture group and the control group is shown in figure 1, and the statistical analysis difference is significant (p < 0.01). As can be known from figure 1 (the horizontal coordinate is culture time), the medium provided by the embodiment of the invention can improve the cell viability.
In addition, L ewis (lung cancer cells) cultured in a conventional medium containing a basal medium was used as a control group, L ewis cultured in the medium provided in example 5 was used as an oxygen carrier culture group, and cells cultured in the control group and the oxygen carrier culture group were subjected to a soft agarose colony formation assay, specifically, 7 × 103Inoculating L ewis cells to 6-well plate containing the above culture medium, culturing for 24h to obtain single cell suspension, preparing 1.5% and 0.8% agarose solution, mixing 1.5% agarose with two times of DMEM medium, pouring into 5cm dish, cooling at room temperature, mixing 0.8% agarose solution with two times of DMEM medium, adding single cell suspension, mixing, pouring into 1.5% agarose plate, and culturing at 37 deg.C and 5% CO2Cultured in an incubator and counted under a microscope as shown in FIG. 2. As can be seen from FIG. 2, the culture medium provided by the example of the present invention has a large number of cells.
In summary, the embodiment of the present invention covers the hydrogel layer on the oxygen carrier, which is beneficial to prevent the oxygen carrier from possibly having toxic effect on cells, and also enables oxygen to permeate into the culture solution through the hydrogel layer, so that the distribution is more uniform, thereby being beneficial to the culture of cells. In addition, the linear continuous oxygen release capacity of the perfluorocarbon compound adopted by the embodiment of the invention is matched with the s-shaped oxygen release capacity and the capacity of maintaining a certain oxygen concentration of the artificial red blood cells and the modified hemoglobin solution, so that the requirements of different oxygen concentration supplies in different periods can be met, and the culture requirements under different conditions can be customized.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for preparing a substrate material of a culture medium, comprising the steps of:
after the oxygen carrier is subjected to disinfection and sterilization and oxygen exposure treatment, adding a hydrogel solution for covering treatment to obtain the base substance; the oxygen carrier is one or more of perfluorocarbon compounds, artificial red blood cells and modified hemoglobin solution.
2. The method for preparing a substrate material of a culture medium according to claim 1, wherein the hydrogel in the hydrogel solution is one or more of agarose, collagen, alginate, hyaluronic acid, methacrylate gelatin, polyethylene glycol and cellulose.
3. The method for preparing the substrate material of the culture medium according to claim 2, wherein the hydrogel solution has a hydrogel mass concentration of 2% to 5%.
4. The method of claim 1, wherein the perfluorocarbon compound is one or more selected from the group consisting of perfluorodecalin, perfluorotributylamine, perfluorotripropylamine, perfluoro-N- (4-methylcyclohexyl) piperidine and perfluorobromoalkane.
5. The method of claim 1, wherein the modified hemoglobin solution is an organic cross-linked modified hemoglobin solution; the organic matter is one of raffinose, polyethylene glycol, disuccinimidyl suberate and glutaraldehyde.
6. The method for preparing a substrate material of a culture medium according to claim 1, wherein the gas for the oxygen exposure treatment is a mixed gas of oxygen and carbon dioxide, and the pressure for the oxygen exposure treatment is 0.08 to 0.12 MPa.
7. The method of claim 6, wherein the volume ratio of oxygen to carbon dioxide is (93-97) to (3-7).
8. A substrate obtained by the production method according to any one of claims 1 to 6.
9. A culture medium comprising the substrate material of claim 8.
10. Use of a medium according to claim 9 for the cultivation of animal cells.
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| WO2007094511A1 (en) * | 2006-02-15 | 2007-08-23 | Oxygenix Co., Ltd. | Animal cell culture method using hemoglobin-based oxygen carrier |
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| CN103251980A (en) * | 2013-05-17 | 2013-08-21 | 中国人民解放军第四军医大学 | Preparation method of perfluorotributylamine-fibrous protein hydrogel compounded nerve conduit |
| CN105749257A (en) * | 2016-01-25 | 2016-07-13 | 四川大学华西医院 | Hemoglobin type oxygen-carrying nano gel and preparation method and application thereof |
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2020
- 2020-05-15 CN CN202010414203.0A patent/CN111411070A/en not_active Withdrawn
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| WO2007094511A1 (en) * | 2006-02-15 | 2007-08-23 | Oxygenix Co., Ltd. | Animal cell culture method using hemoglobin-based oxygen carrier |
| CN101381693A (en) * | 2008-10-14 | 2009-03-11 | 陈祥海 | Controllable high-efficiency microorganism growth medium/liquid and preparation method thereof |
| US20110306581A1 (en) * | 2008-12-08 | 2011-12-15 | University Of Utah Research Foundation | Stable perfluorocarbon emulsion for use as an artificial oxygen carrier |
| CN103251980A (en) * | 2013-05-17 | 2013-08-21 | 中国人民解放军第四军医大学 | Preparation method of perfluorotributylamine-fibrous protein hydrogel compounded nerve conduit |
| CN105749257A (en) * | 2016-01-25 | 2016-07-13 | 四川大学华西医院 | Hemoglobin type oxygen-carrying nano gel and preparation method and application thereof |
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Application publication date: 20200714 |