CN111320779A - Method for modifying surface of substrate and cell culture apparatus - Google Patents
Method for modifying surface of substrate and cell culture apparatus Download PDFInfo
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Abstract
The invention discloses a substrate surface modification method and a cell culture device. The method for modifying the surface of the base material comprises the following steps: s1, first ion treatment: initiating gas to carry out plasma etching treatment on the surface of the base material under the action of discharge so as to form surface free radicals on the surface of the base material; s2, second ion treatment: carrying out secondary ion treatment on the substrate by taking the surface free radicals as initiating substances; s3, graft polymerization treatment: graft-polymerizing a graft polymerization reaction monomer to the surface of the substrate treated in step S2 to introduce a target functional group. The cell culture device comprises a cell culture scaffold, and the cell culture scaffold is modified by the substrate surface modification method. The substrate surface modification method can effectively improve the surface hydrophilic performance, broaden the application range and field of products, realize serum-free culture to reduce the cost of users and is easy to realize industrialization.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a substrate surface modification method and a cell culture device.
Background
A method of improving the properties of the surface of a plastic article by physical or chemical means is called surface modification. The main common methods for modifying the surface of the high polymer material comprise physical modification methods such as surface mechanical modification, surface coating modification, surface vacuum plating, sputtering, spraying and the like; flame modification, solution treatment, discharge, ray irradiation, ion plating, electroplating, graft polymerization modification and other chemical modification methods. Most cells in animals and human bodies, such as fibroblasts, skeletal tissues (bones and cartilages), cardiac muscle, smooth muscle, liver, lung, kidney, mammary gland skin glial cells and the like, grow in an adherent manner when being placed in vitro for culture in vitro, so that the cell surface of an in vitro cell culture device is required to have certain hydrophilicity, and the requirement of adherent cell in vitro culture can be met.
At present, in the field of consumable materials in biological laboratories, the corona discharge modification technology and the plasma treatment polymer material surface modification technology are generally adopted to carry out hydrophilic modification on the material surface, so that the surface tension of the material is increased, and the cell adhesion performance is improved. Corona discharge modification technology and plasma treatment method for modifying the surface of high molecular material are generally initiated by atmosphere or single gas, the thickness of the surface modification layer is extremely thin (from a few nanometers to hundreds of nanometers), and free radicals are formed on the surface or new atoms or groups are introduced to make the surface of the material hydrophilic or other biocompatible. However, due to the instability of the reaction depth and the introduced groups, the two common modification techniques are very easy to cause that the hydrophilic surface is not stable enough and the hydrophilicity is easy to degrade, and the hydrophilicity is gradually weakened along with the increase of the storage time of the high polymer material product. The hydrophilicity of the hydrophilic surface prepared by the two technologies reaches a range of 40-60 degrees by measuring a water contact angle (the smaller the contact angle is, the larger the surface tension is, the stronger the performance of adhering other substances on the surface is), the in vitro culture of the adherent cells with good adherence performance can be basically met, but the requirements of neuron cells, stem cells, primary cells and the like on the in vitro culture conditions cannot be met, the requirement on the surface adhesion of a culture device is higher, and the requirements of scientific research and clinical application of cell culture under the conditions of low serum and no serum for eliminating the interference of the serum cannot be met.
Disclosure of Invention
Therefore, it is necessary to provide a substrate surface modification method and a cell culture apparatus, which can effectively improve surface hydrophilicity, broaden the application range and field of products, realize serum-free culture to reduce user cost, and facilitate industrialization.
A method for modifying the surface of a substrate, comprising the steps of:
s1, first ion treatment: initiating gas to carry out plasma etching treatment on the surface of the base material under the action of discharge so as to form surface free radicals on the surface of the base material;
s2, second ion treatment: carrying out plasma etching treatment on the substrate by taking the surface free radicals as initiating substances;
s3, graft polymerization treatment: graft-polymerizing a graft polymerization reaction monomer to the surface of the substrate treated in step S2 to introduce a target functional group so that the surface of the substrate forms a superhydrophilic cell growth surface having a contact angle to water of 2 ° to 8 °.
In one embodiment, the initiating gas in step S1 is one or a combination of oxygen, nitrogen and inert gas.
In one embodiment, the initiating gas in step S1 is Ar and O in a volume ratio of 1:12And (4) combining.
In one embodiment, the graft polymerization monomer is a compound that itself can provide an oxygen-containing group or that can react with the substrate material by breaking a chemical bond to form an oxygen-containing group.
In one embodiment, the oxygen-containing group is a carbonyl group, a hydroxyl group, or a carboxyl group.
In one embodiment, the process conditions in the first ion treatment in step S1 are as follows:
the vacuum degree is 20mT-150mT, the temperature is 30-55 ℃, the gas flow is 5sccm-80sccm, the processing power is 60w-350w, and the processing time is 100s-1200 s.
In one embodiment, the process conditions in the first ion treatment in step S1 are as follows: the vacuum degree is 20mT-50mT, the temperature is 30-35 ℃, the gas flow is 20sccm-50sccm, the processing power is 100w-250w, and the processing time is 300s-800 s.
In one embodiment, the process conditions in the second ion treatment in step S2 are as follows:
the vacuum degree is 20mT-150mT, the temperature is 30-55 ℃, the gas flow is 20sccm-180sccm, the processing power is 60w-350w, and the processing time is 100s-1200 s.
In one embodiment, the process conditions in the second ion treatment in step S2 are as follows:
the vacuum degree is 50mT-100mT, the temperature is 40-50 ℃, the gas flow is 140sccm-160sccm, the processing power is 100w-200w, and the processing time is 500s-800 s.
A cell culture device comprising a cell culture scaffold, said cell culture scaffold being modified by said substrate surface modification process to obtain a superhydrophilic cell growth surface.
The substrate surface modification method can effectively improve the surface hydrophilic property, broaden the application range and field of products, realize serum-free culture to reduce the cost of users and is easy to realize industrialization. By adopting the substrate surface modification method, the hydrophilicity of the substrate surface is enhanced, the contact angle is below 5 degrees, and after 3 months of accelerated aging test, the hydrophilicity of the substrate surface does not decline, and in addition, cells which are difficult to be cultured by adherence in vitro can grow well on the basic surface treated by the method, and the low serum and serum-free culture of conventional cells and cells which are difficult to be cultured can be met.
Drawings
FIG. 1 is a schematic diagram of a method for modifying the surface of a substrate according to examples 1 to 3 of the present invention;
FIG. 2 is a schematic view of contact angles of different treatment methods on the surface of the polystyrene material substrate in example 5;
FIG. 3 is an infrared spectrum of a surface of a polystyrene substrate in example 5 according to various treatment methods;
FIG. 4 is an atomic force microscope surface topography analysis chart of three different treatment methods for the surface of the polystyrene material substrate in example 5;
FIG. 5 is a graph showing the aging test of conventional hydrophilic pretreatment and super-hydrophilic treatment on the surface of a polystyrene substrate in example 5;
FIG. 6 is a graph showing the comparison between cell attachment and proliferation of the polystyrene substrate of example 5 after three different treatments;
FIG. 7 is a third experimental in vitro cell growth assay of the product of example 5 after treatment with the substrate surface modification method of the present invention;
FIG. 8 is ｃA culture observation of 48h LNCaP-A cells and EVC304 cells at ｃA serum concentration of 2% in example 5;
FIG. 9 is a 48h culture comparison experiment chart of semi-adherent cells 293T after three different treatment methods on the surface of the polystyrene material substrate in example 5.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1
The present example provides a method for modifying a surface of a substrate.
A method for modifying the surface of a substrate, comprising the steps of:
s1, first ion treatment: and carrying out plasma etching treatment on the surface of the base material by the initiating gas under the action of electric discharge so as to form surface free radicals on the surface of the base material. The initiating gas is one or a combination of oxygen, nitrogen and inert gas.
The process conditions for the first ion treatment were as follows: the vacuum degree is 20mT-150mT, the temperature is 30-55 ℃, the gas flow is 5sccm-80sccm, the processing power is 60w-350w, and the processing time is 100s-1200 s.
S2, second ion treatment: and carrying out secondary ion treatment on the substrate by taking the surface free radicals as initiating substances.
The process conditions for the second ion treatment were as follows: the vacuum degree is 20mT-150mT, the temperature is 30-55 ℃, the gas flow is 20sccm-180sccm, the processing power is 60w-350w, and the processing time is 100s-1200 s.
S3, graft polymerization treatment: graft-polymerizing a graft polymerization reaction monomer to the surface of the substrate treated in step S2 to introduce a target functional group so that the surface of the substrate forms a superhydrophilic cell growth surface having a contact angle to water of 2 ° to 8 °. The grafting polymerization reaction monomer is a compound which can provide an oxygen-containing group or can generate the oxygen-containing group by chemical bond breaking and re-reaction with a substrate material. The oxygen-containing group is a carbonyl group, a hydroxyl group or a carboxyl group.
The substrate surface modification method can effectively improve the surface hydrophilic property, broaden the application range and field of products, realize serum-free culture to reduce the cost of users and is easy to realize industrialization. By adopting the substrate surface modification method, the hydrophilicity of the substrate surface is enhanced, the contact angle is below 5 degrees, and after 3 months of accelerated aging test, the hydrophilicity of the substrate surface does not decline, and in addition, cells which are difficult to be cultured by adherence in vitro can grow well on the basic surface treated by the method, and the low serum and serum-free culture of conventional cells and cells which are difficult to be cultured can be met.
Example 2
The present example provides a method for modifying a surface of a substrate.
A method for modifying the surface of a substrate, comprising the steps of:
s1, first ion treatment: introducing an initiating gas into the plasma reaction chamber and carrying out plasma etching treatment on the surface of the base material under the action of discharge so as to form surface free radicals on the surface of the base material. The initiating gas is one or a combination of oxygen, nitrogen and inert gas.
The process conditions for the first ion treatment were as follows: the vacuum degree is 20mT-50mT, the temperature is 30-35 ℃, the gas flow is 20sccm-50sccm, the processing power is 100w-250w, and the processing time is 300s-800 s.
S2, second ion treatment: and carrying out secondary ion treatment on the substrate by taking the surface free radicals as initiating substances.
The process conditions for the second ion treatment were as follows: the vacuum degree is 50mT-100mT, the temperature is 40-50 ℃, the gas flow is 140sccm-160sccm, the processing power is 100w-200w, and the processing time is 500s-800 s.
S3, graft polymerization treatment: graft-polymerizing a graft polymerization reaction monomer to the surface of the substrate treated in step S2 to introduce a target functional group so that the surface of the substrate forms a superhydrophilic cell growth surface having a contact angle to water of 2 ° to 8 °. The grafting polymerization reaction monomer is a compound which can provide an oxygen-containing group or can generate the oxygen-containing group by chemical bond breaking and re-reaction with a substrate material. The oxygen-containing group is a carbonyl group, a hydroxyl group or a carboxyl group.
Example 3
The present example provides a method for modifying a surface of a substrate.
Experiment one
A method for modifying the surface of a substrate, comprising the steps of:
s1, first ion treatment: selecting four cell culture dishes with the diameter of 10cm as a base material, introducing an initiating gas into a plasma reaction chamber, and performing plasma etching treatment under the action of discharge so as to form surface free radicals on the surface of the base material. The initiating gas is a combination of oxygen and nitrogen, and the volume ratio of the oxygen to the nitrogen is 1: 1.
The process conditions for the first ion treatment were as follows: the vacuum degrees of the four substrates are respectively 10mT, 20mT, 50mT and 100mT, the temperature is 40 ℃, the gas flow is 250sccm, the processing power is 250w, and the processing time is 300 s.
S2, second ion treatment: and carrying out secondary ion treatment on the substrate by taking the surface free radicals as initiating substances. The process conditions for the second ion treatment were as follows: the vacuum degrees of the four substrates are respectively 10mT, 20mT, 50mT and 100mT, the temperature is 32 ℃, the gas flow is 250sccm, the processing power is 100w, and the processing time is 600 s.
S3, graft polymerization treatment: and graft-polymerizing an acrylic monomer to the surface of the substrate treated in step S2 to introduce a target functional group so that the surface of the substrate forms a superhydrophilic cell growth surface. A schematic diagram of the substrate surface modification process is shown in fig. 1.
The method comprises the steps of taking distilled water as a test solution, performing static measurement by using a contact angle tester, testing 5 points of each sample, taking the average value of the 5 points as a test result, testing the contact angle of the treated product in a constant temperature and constant humidity state, respectively performing tracking measurement on the contact angle after the sample is placed for 7 days, and researching the aging change of the sample after the first low-temperature ion treatment.
The data of the test shows that the contact angle of a 10cm cell culture dish is gradually reduced along with the reduction of the vacuum value, the contact angle is the lowest at 20mT and reaches the minimum value of 2 degrees, when the vacuum degree is reduced to 10mT, the surface contact angle is not reduced any more, a sample after being placed for 7 days is monitored, the surface contact angle is found to be slightly changed, the contact angle is increased to 8 degrees from the original 2 degrees, but the contact angle is continuously stabilized at the value after continuous detection for several weeks.
Experiment two
A method for modifying the surface of a substrate, comprising the steps of:
s1, first ion treatment: selecting a plurality of cell culture dishes with the diameter of 10cm and made of polystyrene as a base material, introducing an initiating gas into a plasma reaction chamber, and carrying out plasma etching treatment on the surface of the base material under the action of discharge so as to form surface free radicals on the surface of the base material. The initiating gas is a combination of oxygen and nitrogen, and the volume ratio of the oxygen to the nitrogen is 1: 1.
The process conditions for the first ion treatment were as follows: the vacuum degree is 20mT, the temperature is 32 ℃, the processing power is 1000w, and the processing time is 600 s. The gas flow rate for several substrates was gradually increased from 0 to 300 sccm.
S2, second ion treatment: and carrying out secondary ion treatment on the substrate by taking the surface free radicals as initiating substances. The process conditions for the second ion treatment were as follows: the vacuum degree is 20mT, the temperature is 32 ℃, the processing power is 1000w, and the processing time is 600 s. The gas flow rate for several substrates was gradually increased from 0 to 300 sccm.
S3, graft polymerization treatment: and graft-polymerizing an acrylic monomer to the surface of the substrate treated in step S2 to introduce a target functional group so that the surface of the substrate forms a superhydrophilic cell growth surface.
The method comprises the steps of taking distilled water as a test solution, performing static measurement by using a contact angle tester, testing 5 points of each sample, taking the average value of the 5 points as a test result, testing the contact angle of the treated product in a constant temperature and constant humidity state, respectively performing tracking measurement on the contact angle after the sample is placed for 7 days, and researching the aging change of the sample after the first low-temperature ion treatment.
The contact angle exhibited a decrease from 45 ° to 2 ° with increasing gas flow, indicating that the higher the gas flow, the more acrylic monomer was grafted and the lower the contact angle, but that the contact angle reached the minimum value of 2 ° when the treatment gas flow was 150 sccm.
Experiment three
A method for modifying the surface of a substrate, comprising the steps of:
s1, first ion treatment: selecting a plurality of cell culture dishes with the diameter of 10cm and made of polystyrene as a base material, introducing an initiating gas into a plasma reaction chamber, and carrying out plasma etching treatment on the surface of the base material under the action of discharge so as to form surface free radicals on the surface of the base material. The initiating gas is a combination of oxygen and nitrogen, and the volume ratio of the oxygen to the nitrogen is 1: 1.
The process conditions for the first ion treatment were as follows: the vacuum degree is 20mT, the temperature is 32 ℃, the gas flow is 150sccm, the processing time is 600s, and the processing power is gradually and gradually changed from 0-1500 w.
S2, second ion treatment: and carrying out secondary ion treatment on the substrate by taking the surface free radicals as initiating substances. The process conditions for the second ion treatment were as follows: the vacuum degree is 20mT, the temperature is 32 ℃, the gas flow is 150sccm, the processing time is 600s, and the processing power is gradually and gradually changed from 0-1500 w.
S3, graft polymerization treatment: and graft-polymerizing an acrylic monomer to the surface of the substrate treated in step S2 to introduce a target functional group so that the surface of the substrate forms a superhydrophilic cell growth surface.
The method comprises the steps of taking distilled water as a test solution, performing static measurement by using a contact angle tester, testing 5 points of each sample, taking the average value of the 5 points as a test result, testing the contact angle of the treated product in a constant temperature and constant humidity state, respectively performing tracking measurement on the contact angle after the sample is placed for 7 days, and researching the aging change of the sample after the first low-temperature ion treatment.
The contact angle shows a decrease from 25 ℃ to 2 ℃ with increasing treatment power over the treatment period, which means that with increasing treatment power, more acrylic monomer is grafted and the contact angle becomes lower, but already reaches a minimum value of 2 ℃ at a treatment power of 1000 w.
Experiment four
A method for modifying the surface of a substrate, comprising the steps of:
s1, first ion treatment: selecting a plurality of cell culture dishes with the diameter of 10cm and made of polystyrene as a base material, introducing an initiating gas into a plasma reaction chamber, and carrying out plasma etching treatment on the surface of the base material under the action of discharge so as to form surface free radicals on the surface of the base material. The initiating gas is a combination of oxygen and nitrogen, and the volume ratio of the oxygen to the nitrogen is 1: 1.
The process conditions for the first ion treatment were as follows: the vacuum degree is 20mT, the temperature is 32 ℃, the gas flow is 150sccm, and the processing power is 1000 w. The treatment time is gradually increased from 0 to 1000 s.
S2, second ion treatment: and carrying out secondary ion treatment on the substrate by taking the surface free radicals as initiating substances. The process conditions for the second ion treatment were as follows: the vacuum degree is 20mT, the temperature is 32 ℃, the gas flow is 150sccm, and the processing power is 1000 w. The treatment time is gradually increased from 0 to 1000 s.
S3, graft polymerization treatment: and graft-polymerizing an acrylic monomer to the surface of the substrate treated in step S2 to introduce a target functional group so that the surface of the substrate forms a superhydrophilic cell growth surface.
The method comprises the steps of taking distilled water as a test solution, performing static measurement by using a contact angle tester, testing 5 points of each sample, taking the average value of the 5 points as a test result, testing the contact angle of the treated product in a constant temperature and constant humidity state, respectively performing tracking measurement on the contact angle after the sample is placed for 7 days, and researching the aging change of the sample after the first low-temperature ion treatment.
The contact angle shows a decrease from 45 ° to 2 ° with an increase in the discharge time, which means that the longer the time, the more acrylic monomer is grafted and the contact angle becomes lower, but the contact angle already reaches the minimum value of 2 ° when the treatment time is 600 seconds.
According to the first experiment and the fourth experiment, the optimal process conditions for determining the pretreatment by combining the grafting of the acrylic monomer, the contact angle and the cell culture effect are as follows: ar and O2The mixed gas is maintained at a constant temperature of 32 degrees under the vacuum degree of 20mT, the gas flow rate is 150sccm, the processing power is 1000w, and the processing time is 600 s.
Example 4
The present example provides a method for modifying a surface of a substrate.
In this example, Ar and Ar/O are used respectively2(volume ratio 1:1) and O2Three gases in polystyrene material base materialThe surface of (2) is graft-polymerized with an acrylic monomer.
The substrate surface modification method described in example 2 was followed, the first group all being treated with 99.999% Ar gas: introducing 99.999% Ar gas into a plasma reaction chamber, carrying out primary ion treatment on a polystyrene material substrate under the conditions of vacuum degree of 20mT, constant temperature of 32 degrees, gas flow rate of 150sccm, treatment power of 1000w and treatment time of 600s, so that a concave-convex structure is formed on the surface of the substrate, a modified acrylic monomer is grafted, an acrylic monomer is grafted on the surface of the substrate, a super-hydrophilic cell growth surface is formed, and SEM and CA analysis shows that the contact angle of the surface of a product can reach 6-15 degrees.
The second group used 99.999% Ar and O2Mixing according to the volume ratio of 50% for treatment: 99.999% of Ar and O2(1: 1) introducing the mixed gas into a plasma reaction chamber, and carrying out primary ion treatment on the polystyrene material substrate under the conditions of vacuum degree of 20mT, constant temperature of 32 degrees, gas flow rate of 150sccm, treatment power of 1000w and treatment time of 600s to enable the surface of the polystyrene material substrate to form a concave-convex structure and graft a modified acrylic monomer, so that the surface of the polystyrene material substrate is grafted with an acrylic monomer to form a super-hydrophilic cell growth surface. SEM and CA analysis show that the contact angle of the product surface can reach 2-8 degrees.
The third group adopts the total of 99.9 percent of O2Gas treatment: mixing 99.9% of O2Introducing gas into a plasma reaction chamber, and carrying out first ion treatment on the polystyrene material substrate under the conditions of vacuum degree of 20mT, constant temperature of 32 degrees, gas flow rate of 150sccm, treatment power of 1000w and treatment time of 600s, so that a concave-convex structure is formed on the surface of the polystyrene material substrate, a modified acrylic monomer is grafted, an acrylic monomer is grafted on the surface of the polystyrene material substrate, and a super-hydrophilic cell growth surface is formed. SEM and CA analysis shows that the contact angle of the product surface can reach 4-10 deg.
Through the mixing mode of three different gases, the method is found that under the same process condition, 99.999 percent of Ar and O are adopted2Product list treated in a mixed manner at 50% by volume of eachThe face contact angle can be minimized.
Example 5
The present example provides a cell culture apparatus.
A cell culture device comprising a cell culture scaffold modified by the substrate surface modification method of example 2. Wherein, the process parameters in the step S1 and the step S2 are Ar and O2The mixed gas (volume ratio 1:1) is at a constant temperature of 32 degrees under a vacuum degree of 20mT, the gas flow rate is 150sccm, the processing power is 1000w, and the processing time is 600 s.
Performance analysis
The contact angle of the surface of the substrate of the acrylic graft-modified polystyrene material was first measured, as shown in fig. 2 a-2 c, fig. 2a (left view) shows an untreated polystyrene substrate, fig. 2b (middle view) shows a direct plasma hydrophilically treated polystyrene substrate, and fig. 2c (right view) shows a treated polystyrene substrate according to the method for modifying the surface of the substrate of the present invention. The surface of the base material of the untreated polystyrene material is hydrophobic, the contact angle is more than 90 degrees, the contact angle can reach 30-45 degrees after one-step direct hydrophilic treatment by plasma, and the contact angle of the surface of the base material of the polystyrene material treated by the base material surface modification method can reach less than 10 degrees, so that the base material surface modification method can greatly improve the hydrophilicity of the surface of the base material.
Referring to FIG. 3 (the abscissa in FIG. 3 is the wavelength and the ordinate is the absorption peak), it can be seen that only a small amount of absorption peaks of water and carbon dioxide in the air and oxygen-containing functional groups caused by partial oxidation occurring during injection molding and long-term contact with air are present on the surface of the untreated polystyrene substrate by infrared spectroscopic analysis of the untreated polystyrene substrate, the plasma hydrophilically treated polystyrene substrate and the substrate surface modification method of the present invention treated polystyrene substrate; the adsorption peak of the oxygen-containing functional group on the surface of the polystyrene substrate after the plasma hydrophilic treatment becomes more obvious; the characteristic absorption peaks of hydroxyl, carbonyl and carboxyl of the polystyrene substrate treated by the substrate surface modification method are more obvious, and the peak area is larger, namely the number of oxygen-containing functional groups on the surface of the acrylic acid grafted polystyrene substrate is more abundant than that on the surface of the conventional hydrophilic treated polystyrene substrate. X polystyrene photoelectron spectroscopy scans of the O element and the C element on the surface of the untreated polystyrene substrate, the plasma hydrophilically treated polystyrene substrate and the polystyrene substrate treated by the substrate surface modification method of the present invention, and the analysis results are shown in Table 1.
TABLE 1 photoelectron spectroscopy analysis results of different treatment modes of the surface of polystyrene substrate
As can be seen from Table 1, the surface of the untreated polystyrene substrate is almost entirely carbon, and only a small amount of oxygen, which is adsorbed or undergoes surface oxidation, exists; the oxygen element content of the surface of the polystyrene base material after the conventional hydrophilic treatment is increased to 5.18 percent, which shows that the surface is partially oxidized after the first ion treatment, and oxygen-containing groups such as hydroxyl, carbonyl and the like are introduced; after the surface is subjected to acrylic acid plasma grafting treatment, the oxygen element content on the surface of the polystyrene substrate is increased sharply and reaches 26.42%, hydroxyl groups, carbonyl groups and a large number of carboxyl functional groups exist, and the analysis results are consistent with the contact angle measurement, infrared spectroscopy and X polystyrene surface element analysis results.
By atomic force microscopy analysis of the surfaces of untreated polystyrene substrates, plasma hydrophilically treated polystyrene substrates, and the polystyrene substrates treated by the substrate surface modification method of the present invention, as shown in FIG. 4, the surfaces of untreated polystyrene substrates were extremely smooth and flat except for the influence of dust particles adsorbed on the surfaces during some sample preparation. The surface becomes rougher after the conventional hydrophilic treatment in the first step, which indicates that the surface of the polystyrene material passes Ar/O2After the first ion treatment, the chemical structure of the material surface is damaged, and a plurality of C-C and C-H bonds are broken and oxidized, so that the material surface appearance shows a plurality of rough pits in a nanometer scale. After the conventional hydrophilic treatment of the surface of the polystyrene material in the first step, the Ar/O in the second step is continuously carried out2After acrylic acid plasma grafting treatment, not only the chemical structure of the surface is destroyed and oxidized, but also acrylic acid graft polymerization is carried out on the basis of the broken chemical structure, so that the surface has hydroxyl and carbonyl functional groups, and a plurality of carboxyl functional groups are introduced by grafting a plurality of acrylic acid polymer side chains, so that the roughness of the surface of the polystyrene material after super-hydrophilic treatment is changed rapidly from the aspect of appearance, and not only a plurality of pits are formed, but also a plurality of grafted polyacrylic acid nano-particles are fixed on the surface.
Conventional hydrophilically treated polystyrene substrates and treated polystyrene substrates of the present invention were placed in a constant temperature and humidity chamber at 60 ℃ according to the accelerated aging test standard for materials of ASTM F1980, and then the contact angle values of the materials with water were measured at intervals. As shown in fig. 5 (abscissa is time, ordinate is contact angle in fig. 5), the contact angle of the conventional hydrophilically treated polystyrene substrate surface with water rapidly increases from 30 ° to 60 ° 3 weeks before aging under the constant temperature and humidity condition at 60 ℃, and then increases relatively slowly with the increase of aging time, and the contact angle is substantially maintained at about 65 °, because oxygen-containing functional groups generated on the polystyrene substrate surface etched by pure plasma gas are still in an unstable high-energy state at first, and the unstable functional groups in the high-energy state rapidly escape and are converted into stable high-energy groups with the increase of aging time, and finally, only the stable oxygen-containing functional groups are remained on the surface. Under the same aging condition, the contact angle of the super-hydrophilic polystyrene substrate surface treated by the acrylic acid plasma grafting reaction of the invention with water rises very slowly from the initial 5 degrees and is basically kept below 10 degrees, because most of the surface of the polystyrene substrate treated by the invention is polyacrylic acid graft polymer connected by covalent bonds and is not oxygen-containing groups in an unstable high-energy state, the contact angle of the surface is basically maintained unchanged, and the trend that the contact angle slightly rises is caused by gradual and slow separation of part of the oxygen-containing functional groups in the high-energy state occurs. Therefore, the surface of the base material prepared by the base material surface modification method provided by the invention not only has richer oxygen-containing groups and extremely low contact angle, but also can be permanently and stably anchored on the surface of the base material by the oxygen-containing groups, and the surface contact angle can be kept below 10 degrees in the shelf life of 3 years.
As shown in FIG. 6, the results of cell adhesion and proliferation comparative experiments were conducted after culturing 3T3 cells for 48 hours in FBS serum concentration of 10%, wherein the untreated polystyrene substrate (left), the plasma hydrophilically treated polystyrene substrate (middle), and the substrate surface modification method of the present invention treated polystyrene substrate (right). It can be seen that the cells attached to the surface of the untreated polystyrene substrate have very low cell attachment and proliferation ability, and the morphology of the cells is also very poor. When the surface of the polystyrene substrate is subjected to hydrophilic pretreatment, the cell adherence and proliferation capacity of the surface of the polystyrene substrate are obviously improved, the cell morphology becomes more regular, part of the polystyrene substrate is stretched, and part of the polystyrene substrate is still in a spherical state. However, the adherence and proliferation capacity of the polystyrene substrate material cells treated by the substrate material surface modification method are greatly improved compared with the conventional hydrophilic pretreatment surface, the cells are very spread in shape and almost all are in a regular fusiform shape, so that the original functional expression of the cells is more facilitated, and the substrate material prepared by the substrate material surface modification method is more favorable for adherence and proliferation of the cells compared with the substrate material applied on a large scale in the market at present.
As shown in fig. 7, a third detection report of an in vitro cell growth experiment of a substrate product prepared by the substrate surface modification method of the present invention shows that L929 cells are almost completely attached after 4h of inoculation, the cells are completely attached after 8h, the cells are significantly proliferated after 48h, and the cells in different culture media have slightly different morphologies but are in a stretched fusiform. Therefore, the substrate surface cells prepared by the substrate surface modification method have strong adherence and proliferation, have no cytotoxicity and are very suitable for in vitro adherence culture of the cells.
FIG. 8 is ｃA photograph of ｃA 48h culture observation of LNCaP-A cells and EVC304 cells at ｃA serum concentration of 2%, wherein the left image is 2% FBS and the LNCaP-A cells are cultured for 48 h; the right panel is 2% FBS, EVC304 cells in 48h culture. Therefore, the cells can still realize good adherence and proliferation under low serum concentration, which shows that the base material product obtained by the base material surface modification method is completely suitable for cell culture under the low serum condition, different cells show different adherence forms, and the low serum culture is favorable for reducing the culture cost of the cells.
As shown in fig. 9, a 48h culture comparison experiment chart of semi-adherent cells 293T after three different treatment modes on the surface of the polystyrene material substrate is shown, wherein the untreated polystyrene substrate (left), the plasma hydrophilically treated polystyrene substrate (middle), and the substrate surface modification method of the present invention are used for treating the polystyrene substrate (right). The semi-adherent cell 293T is cultured for 48h under the serum concentration of 10 percent, and the cells on the surface of the untreated polystyrene substrate are hardly adherent and do not proliferate; the cells on the surface of the polystyrene substrate subjected to conventional hydrophilic treatment have weak adherence and proliferation capacity; the adherence and proliferation capacities of the polystyrene substrate surface cells treated by the substrate surface modification method are strong, and the polystyrene substrate treated by the substrate surface modification method can completely meet the culture of semi-adherence and other refractory cells. Compared with the conventional cell culture surface product, the substrate product obtained by the substrate surface modification method can greatly meet the requirement of the culture of the cells difficult to culture.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
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 modifying the surface of a substrate, comprising the steps of:
s1, first ion treatment: initiating gas to carry out plasma etching treatment on the surface of the base material under the action of discharge so as to form surface free radicals on the surface of the base material;
s2, second ion treatment: carrying out plasma etching treatment on the substrate by taking the surface free radicals as initiating substances;
s3, graft polymerization treatment: graft-polymerizing a graft polymerization reaction monomer to the surface of the substrate treated in step S2 to introduce a target functional group so that the surface of the substrate forms a superhydrophilic cell growth surface having a contact angle to water of 2 ° to 8 °.
2. The method of claim 1, wherein the initiating gas in step S1 is one or more of oxygen, nitrogen, and inert gas.
3. The method of claim 2, wherein the initiation gas in step S1 is Ar and O in a volume ratio of 1:12And (4) combining.
4. The method of claim 1, wherein the graft polymerization monomer is a compound that itself can provide an oxygen-containing group or that can generate an oxygen-containing group by a chemical bond cleavage reaction with the substrate material.
5. The method of claim 4, wherein the oxygen-containing group is a carbonyl group, a hydroxyl group, or a carboxyl group.
6. The method for modifying the surface of a substrate according to any one of claims 1 to 5, wherein the process conditions in the first ion treatment in step S1 are as follows:
the vacuum degree is 20mT-150mT, the temperature is 30-55 ℃, the gas flow is 5sccm-80sccm, the processing power is 60w-350w, and the processing time is 100s-1200 s.
7. The method for modifying the surface of a substrate according to claim 6, wherein the process conditions in the first ion treatment in step S1 are as follows: the vacuum degree is 20mT-50mT, the temperature is 30-35 ℃, the gas flow is 20sccm-50sccm, the processing power is 100w-250w, and the processing time is 300s-800 s.
8. The method for modifying the surface of a substrate according to any one of claims 1 to 5, wherein the process conditions in the second ion treatment in step S2 are as follows:
the vacuum degree is 20mT-150mT, the temperature is 30-55 ℃, the gas flow is 20sccm-180sccm, the processing power is 60w-350w, and the processing time is 100s-1200 s.
9. The method for modifying the surface of a substrate according to claim 8, wherein the process conditions in the second ion treatment in step S2 are as follows:
the vacuum degree is 50mT-100mT, the temperature is 40-50 ℃, the gas flow is 140sccm-160sccm, the processing power is 100w-200w, and the processing time is 500s-800 s.
10. A cell culture device comprising a cell culture scaffold modified by the method of substrate surface modification of any one of claims 1 to 9 to obtain a superhydrophilic cell growth surface.
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| CN112851995A (en) * | 2021-01-29 | 2021-05-28 | 苏州赛普生物科技有限公司 | Surface hydrophilic treatment method for cell culture dish |
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| CN116145424A (en) * | 2023-02-23 | 2023-05-23 | 同腾新创(苏州)科技有限公司 | Long-acting surface modified carrier for promoting cell adhesion |
| CN116492505A (en) * | 2023-05-11 | 2023-07-28 | 山东第一医科大学附属眼科医院(山东省眼科医院) | Artificial cornea endothelial transplanting sheet and application thereof |
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| WO2021169599A1 (en) * | 2020-02-28 | 2021-09-02 | 广州洁特生物过滤股份有限公司 | Substrate surface modification method and cell culture apparatus |
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| CN115491307A (en) * | 2022-10-27 | 2022-12-20 | 同腾新创(苏州)科技有限公司 | PET (polyethylene terephthalate) film for cell culture in cell and gene therapy and application |
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| CN116145424A (en) * | 2023-02-23 | 2023-05-23 | 同腾新创(苏州)科技有限公司 | Long-acting surface modified carrier for promoting cell adhesion |
| CN116145424B (en) * | 2023-02-23 | 2024-01-09 | 同腾新创(苏州)科技有限公司 | Long-acting surface modified carrier for promoting cell adhesion |
| CN116492505A (en) * | 2023-05-11 | 2023-07-28 | 山东第一医科大学附属眼科医院(山东省眼科医院) | Artificial cornea endothelial transplanting sheet and application thereof |
| CN116492505B (en) * | 2023-05-11 | 2023-12-08 | 山东第一医科大学附属眼科医院(山东省眼科医院) | Artificial cornea endothelial transplanting sheet and application thereof |
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Application publication date: 20200623 |