CN112480750A - Super-hydrophilic coating for cell culture and preparation method thereof - Google Patents
Super-hydrophilic coating for cell culture and preparation method thereof Download PDFInfo
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Abstract
The invention provides a super-hydrophilic coating for cell culture and a preparation method thereof, wherein the polyvinyl alcohol-chitosan interpenetrating network super-hydrophilic coating is coated on a cell culture device in a spraying, dip-coating or roll-coating mode and the like, and has a lasting and stable super-hydrophilic effect, and the water contact angle of the super-hydrophilic coating is lower than 10 degrees; the prepared cell culture device has strong coating adhesion, high transparency, ultraviolet resistance, water soaking resistance, good biocompatibility, low cytotoxicity and high cell adherence rate.
Description
Technical Field
The invention belongs to the field of biological materials, and particularly relates to a super-hydrophilic coating for cell culture and a preparation method thereof.
Background
Modern biomedical science has advanced rapidly and stem cell culture has become a fundamental work of great importance in many fields of research and application. Conventionally, a culture plate or a culture flask made of polystyrene has been widely used as a tool for cell culture. The polystyrene has good light transmission performance, better strength and plasticity and no toxicity, and becomes the preferred material of disposable cell culture consumables such as disposable cell culture dishes, cell culture plates and the like. However, the polystyrene surface is hydrophobic, so that the polystyrene surface needs to be modified to become hydrophilic so as to be suitable for cell culture.
The TC treatment (Tissue culture treated) is the most common surface hydrophilic modification treatment method for polystyrene culture dishes, and a plasma surface treatment technology is generally adopted. The hydrophilic contact angle of the hydrophilic surface formed by modifying polystyrene by plasma is 60 degrees, and the hydrophilic contact angle is required to be less than 10 degrees for the good adherent growth of the cells, so that the adherent growth of the cells with harsh growth conditions is difficult to meet. Patent 201510194152.4 describes the preparation of a superhydrophilic cell growth surface by plasma techniques.
The nano coating has mild technical conditions and simple process, and the coating thickness can be accurately controlled through the coating amount. The coating technology is simple to operate, expensive equipment is not needed, but the super-hydrophilic coating is poor in stability, low in transparency, low in adhesive force and easy to fall off under the influence of ultraviolet rays and moisture. Meanwhile, the problems of biocompatibility, cytotoxicity, low cell adherence and the like exist, so that the preparation of the super-hydrophilic surface for cell culture based on the nano-coating method is still a great problem.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a super-hydrophilic coating for cell culture and a preparation method thereof, wherein the super-hydrophilic coating has a durable and stable super-hydrophilic effect, and the water contact angle of the super-hydrophilic coating is lower than 10 degrees. Meanwhile, the coating has strong adhesive force, high transparency, ultraviolet resistance, water immersion resistance, good biocompatibility, low cytotoxicity and high cell adherence rate.
In order to achieve the purpose, the invention provides the following technical scheme:
a super-hydrophilic coating for cell culture is a polyvinyl alcohol-chitosan interpenetrating network super-hydrophilic coating, and the water contact angle is 2-8 degrees.
Specifically, the coating is applied to the cell culture device by spraying, dipping, rolling, or the like.
Specifically, the thickness of the coating is 8-30 μm.
The preparation method of the polyvinyl alcohol-chitosan interpenetrating network super-hydrophilic coating comprises the following steps:
1) dissolving chitosan (the viscosity is 400mPa.s) in an acidic aqueous solution, wherein the mass fraction of the chitosan is 1%, and the pH value of the solution is 4-6;
2) adding polyvinyl alcohol 1788 into the solution, heating to 60-80 ℃ for dissolving, wherein the mass ratio of the polyvinyl alcohol to the chitosan is 1: 4-1: 2;
3) adding a small amount of cross-linking agent, heating to 40-80 ℃ and reacting for 1-3 h.
Specifically, the acidic aqueous solution in the step 1) comprises one or more of acetic acid aqueous solution, acrylic acid aqueous solution and HCl aqueous solution;
specifically, the cross-linking agent in the step 3) comprises one or more of glutaraldehyde, glycol and oxalic acid.
The light transmittance of the polystyrene culture dish containing the super-hydrophilic coating prepared by the scheme reaches 95-98 percent;
the polystyrene culture dish containing the super-hydrophilic coating prepared by the scheme has a water contact angle of 2-8 degrees measured by a water contact angle measuring instrument;
the polystyrene culture dish containing the super-hydrophilic coating prepared by the scheme is soaked in pure water for 7 days, the coating does not fall off, and the water contact angle is not attenuated;
the polystyrene culture dish containing the super-hydrophilic coating prepared by the scheme is soaked in an acetic acid solution for 7 days, the coating does not fall off, and the water contact angle is not attenuated;
the polystyrene culture dish containing the super-hydrophilic coating prepared by the scheme is soaked in a sodium hydroxide aqueous solution for 7 days, the coating does not fall off, and the water contact angle does not attenuate.
The super-hydrophilic coating has an adhesive force of 0 grade measured by a hundred-grid test;
after the super-hydrophilic coating is subjected to a high-temperature accelerated aging test, the coating does not fall off after being heated to 80 ℃ for 5000 hours, and the water contact angle is not attenuated;
after the ultra-hydrophilic coating is subjected to an ultraviolet aging test, the coating does not fall off after being subjected to ultraviolet irradiation for 1000 hours, and the water contact angle is not attenuated;
the surface of the super-hydrophilic coating is positively charged, and the zeta potential is 28.6 +/-0.2 mV-30.4 +/-0.2 mV;
the components of the polyvinyl alcohol-chitosan interpenetrating network super-hydrophilic coating are all polymers with good biocompatibility.
The invention also provides a cell culture device, which comprises a polystyrene substrate and the super-hydrophilic coating for cell culture.
Specifically, the polystyrene base layer is one of a culture dish, a culture plate and a culture bottle.
The invention also provides a method for cell culture, which is to plant cells on the cell culture device for culture.
The cells used in the scheme are cells which are difficult to nourish, such as mouse embryo fibroblasts, mouse adipose adult stem cells and the like.
When the culture dish containing the super-hydrophilic coating is used for culturing the cells, the anchorage rate can reach 87-90%, the growth state of the cells is good, and the anchorage rate is only 40-45% when the common TC treatment culture dish is used for treating the anchorage rate.
According to the invention, the chitosan and the polyvinyl alcohol are two single components which are linear molecules, and the formed coating is easy to fall off in an aqueous solution, so that the two hydrophilic polymers are designed into an interpenetrating network structure to form a network structure to enhance the strength of the coating, and meanwhile, the two hydrophilic polymers of the chitosan and the polyvinyl alcohol can be grafted through a chemical reaction, so that the hydrophobic part and the culture dish substrate have good adhesive force, and the hydrophilic groups such as hydroxyl and amino form a super-hydrophilic structure on the outermost surface, so that the adhesive force of the coating is improved, and the substrate can not fall off in water. The amino groups on the surface make the coating positively charged, which facilitates the adsorption of proteins. The coating can be cured at room temperature, is simple and convenient to operate, has low cost and is suitable for large-scale industrial production.
The invention provides a super-hydrophilic coating for cell culture and a preparation method thereof, the super-hydrophilic coating has a low water contact angle of 10 degrees and is positively charged, can ensure that cells which are difficult to culture can grow well adherent, and has good biocompatibility, low cytotoxicity and high cell adherent rate. Meanwhile, the coating has strong adhesive force, high transparency, ultraviolet resistance and water immersion resistance, and has a lasting and stable super-hydrophilic effect.
Drawings
FIG. 1 shows the results of the water contact angle test of the polystyrene culture dish containing the super-hydrophilic coating in example 12;
FIG. 2 is a comparison of anchorage rates of cells cultured on a super hydrophilic coated culture dish and a TC treated culture dish.
Detailed Description
For a better understanding of the present invention, the following further illustrates the present invention with reference to specific examples and drawings, but the present invention is not limited to the following examples.
EXAMPLE 1 preparation of Chitosan solution
1g of chitosan (viscosity 400mPa.s) is dissolved in 99g of acetic acid aqueous solution, the mass fraction of the chitosan is 1 percent, and the pH value of the solution is 4.
Example 2 preparation of a Chitosan solution
1g of chitosan (viscosity 400mPa.s) is dissolved in 99g of acrylic acid aqueous solution, the mass fraction of the chitosan is 1 percent, and the pH value of the solution is 5.
Example 3 preparation of a Chitosan solution
1g of chitosan (viscosity 400mPa.s) is dissolved in 99g of hydrochloric acid solution, the mass fraction of the chitosan is 1 percent, and the pH value of the solution is 6.
Example 4 preparation of Chitosan-polyvinyl alcohol solution
0.25g of polyvinyl alcohol 1788 was added to the solution of example 1 and dissolved by heating to 60 ℃.
Example 5 preparation of Chitosan-polyvinyl alcohol solution
0.4g of polyvinyl alcohol 1788 was added to the solution of example 2 and dissolved by heating to 70 ℃.
Example 6 preparation of Chitosan-polyvinyl alcohol solution
0.5g of polyvinyl alcohol 1788 was added to the solution of example 3 and dissolved by heating to 80 ℃.
Example 7 preparation of polyvinyl alcohol-chitosan interpenetrating network super hydrophilic coating 1
0.05g of glutaraldehyde is added to the solution of example 4, the mixture is heated to 40 ℃ and reacted for 1h, and the resulting superhydrophilic coating has a Zeta potential of 28.6 +/-0.2 mV.
EXAMPLE 8 preparation of polyvinyl alcohol-Chitosan interpenetrating network Superhydrophilic coating 2
0.05g of ethylene glycol was added to the solution of example 5, and the mixture was heated to 60 ℃ to react for 2 hours, and the resulting super-hydrophilic coating was tested to have a Zeta potential of 29.7 mV. + -. 0.1 mV.
Example 9 preparation of polyvinyl alcohol-chitosan interpenetrating network Superhydrophilic coating 3
0.05g of oxalic acid is added into the solution of the example 6, the mixture is heated to 80 ℃ and reacts for 3 hours, and the Zeta potential of the obtained super-hydrophilic coating is tested to be 30.4 +/-0.2 mV.
EXAMPLE 10 culture dish with super-hydrophilic coating
The superhydrophilic coating from example 7 was applied by spraying onto a petri dish at a thickness of 8 μm.
EXAMPLE 11 culture dish with super-hydrophilic coating
The petri dish was soaked in the super-hydrophilic coating obtained in example 8 for 5min and then taken out, the coating thickness being 20 μm.
EXAMPLE 12 Petri dishes with Superhydrophilic coating
The super-hydrophilic coating obtained in example 9 was flow coated onto a polystyrene culture dish by means of a flow coating apparatus, the coating thickness being 30 μm.
The culture dishes containing the super-hydrophilic coatings obtained in example 10, example 11 and example 12 were tested for light transmittance, adhesion, untreated water contact angle, and other treated water contact angles. Wherein the other treatment is as follows:
soaking in pure water for 7 days, removing water, and drying; soaking in 20% acetic acid water solution for 7 days, removing water, and drying; soaking in 10% sodium hydroxide water solution for 7 days, removing water, and drying; high temperature accelerated aging test, heating to 80 ℃ for 5000 hours; and ultraviolet aging test, wherein the ultraviolet irradiation is carried out for 1000 hours.
The test results are shown in the following table:
example 13
The mouse embryonic fibroblast (MEF cell) suspension was centrifuged at 1500rpm for 5 minutes to collect cells, which were washed twice with 30ml of growth medium. The cell pellet was resuspended in 15ml growth medium and added to polystyrene petri dishes treated with super hydrophilic coating and TC, respectively, after which it was cultured in a 37 ℃ cell incubator, after 24 hours fresh growth medium was replaced. After the cells are full, the cells are washed by D-PBS, poured off, digested by pancreatin and passaged according to the ratio of 1: 5. The cells were counted at each passage and observed for adherence using a fluorescence microscope. The cell survival rate of the polystyrene culture dish containing the super hydrophilic coating is more than 95 percent, and the cell adherence rate is 90 percent. The cell viability of the TC-treated polystyrene culture dish was 80% and the cell adherence rate was 40%, and the results are shown in FIG. 2.
Example 14
Similar to example 13, mouse adipose-derived adult stem cells (ADAS cells) were cultured in polystyrene culture dishes with super-hydrophilic coatings, which showed a cell survival rate of 93% or more and a cell adherence rate of 87%, and TC-treated polystyrene culture dishes, respectively. The cell survival rate of the TC-treated polystyrene culture dish was 78%, the cell adherence rate was 45%, and the results are shown in FIG. 2.
Claims (10)
1. The super-hydrophilic coating for cell culture is characterized by being a polyvinyl alcohol-chitosan interpenetrating network super-hydrophilic coating, and the water contact angle is 2-8 degrees.
2. The superhydrophilic coating for cell culture according to claim 1, wherein the coating is applied to the cell culture device by spraying, dipping, rolling or the like.
3. The superhydrophilic coating for cell culture according to claim 1, wherein the coating thickness is 8-30 μ ι η.
4. A method for preparing the super-hydrophilic coating for cell culture according to any one of claims 1 to 3, comprising the steps of:
1) dissolving chitosan in an acidic aqueous solution, wherein the mass fraction of the chitosan is 1%, and the pH value of the solution is 4-6;
2) adding polyvinyl alcohol 1788 into the solution, heating to 60-80 ℃ for dissolving, wherein the mass ratio of the polyvinyl alcohol to the chitosan is 1: 4-1: 2;
3) adding a small amount of cross-linking agent, heating to 40-80 ℃ and reacting for 1-3 h.
5. The method according to claim 4, wherein the acidic aqueous solution in step 1) comprises one or more of an aqueous acetic acid solution, an aqueous acrylic acid solution, and an aqueous HCl solution.
6. The method of claim 4, wherein the step 3) cross-linking agent comprises one or more of glutaraldehyde, ethylene glycol, and oxalic acid.
7. A cell culture device comprising a polystyrene-based layer and the superhydrophilic coating for cell culture of any one of claims 1-3.
8. The cell culture apparatus of claim 7, wherein the polystyrene-based layer is one of a petri dish, a culture plate, and a culture flask.
9. A method for cell culture, characterized in that cells are planted and cultured on the cell culture apparatus according to claim 8.
10. The method for cell culture according to claim 9, wherein the cell is a mouse embryonic fibroblast cell, a mouse adipose adult stem cell.
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| CN114107164A (en) * | 2021-11-02 | 2022-03-01 | 武汉中科先进技术研究院有限公司 | Preparation method of surface for adherent growth of cells in serum-free environment |
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| CN114107164A (en) * | 2021-11-02 | 2022-03-01 | 武汉中科先进技术研究院有限公司 | Preparation method of surface for adherent growth of cells in serum-free environment |
| CN114107164B (en) * | 2021-11-02 | 2024-04-30 | 武汉中科先进材料科技有限公司 | Preparation method of surface for cell wall-attached growth under serum-free condition |
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Application publication date: 20210312 |