CN114920972A - Preparation method of temperature-sensitive culture dish - Google Patents
Preparation method of temperature-sensitive culture dish Download PDFInfo
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- CN114920972A CN114920972A CN202210575187.2A CN202210575187A CN114920972A CN 114920972 A CN114920972 A CN 114920972A CN 202210575187 A CN202210575187 A CN 202210575187A CN 114920972 A CN114920972 A CN 114920972A
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- culture dish
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- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000004140 cleaning Methods 0.000 claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000001035 drying Methods 0.000 claims abstract description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000012535 impurity Substances 0.000 claims abstract description 8
- 238000009832 plasma treatment Methods 0.000 claims abstract description 5
- 239000000758 substrate Substances 0.000 claims abstract description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000007599 discharging Methods 0.000 claims abstract description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 4
- 239000001301 oxygen Substances 0.000 claims abstract description 4
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 4
- 230000010355 oscillation Effects 0.000 claims abstract description 3
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 claims description 37
- 239000004793 Polystyrene Substances 0.000 claims description 8
- 229920002223 polystyrene Polymers 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims 3
- 238000004519 manufacturing process Methods 0.000 claims 1
- -1 isopropyl acryloyl Chemical group 0.000 abstract description 12
- 210000004027 cell Anatomy 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000010559 graft polymerization reaction Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 230000033667 organ regeneration Effects 0.000 description 1
- 125000000864 peroxy group Chemical group O(O*)* 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000010526 radical polymerization reaction Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
- C08J7/12—Chemical modification
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
- C08J7/12—Chemical modification
- C08J7/123—Treatment by wave energy or particle radiation
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
- C08J7/12—Chemical modification
- C08J7/16—Chemical modification with polymerisable compounds
- C08J7/18—Chemical modification with polymerisable compounds using wave energy or particle radiation
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2325/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Derivatives of such polymers
- C08J2325/02—Homopolymers or copolymers of hydrocarbons
- C08J2325/04—Homopolymers or copolymers of styrene
- C08J2325/06—Polystyrene
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Toxicology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention relates to the technical field of culture dish preparation, and particularly discloses a preparation method of a temperature-sensitive culture dish. The preparation method of the temperature-sensitive culture dish comprises the following steps: preparing poly-N isopropyl acryloyl solution from poly-N isopropyl acryloyl water; cleaning a culture dish, removing impurities, and drying to obtain a pretreated culture dish; placing the pretreated culture dish in a plasma treatment device for low-temperature plasma pretreatment; taking out the pretreated substrate after low-temperature plasma pretreatment and exposing the pretreated substrate to the atmospheric environment; placing the poly-N isopropyl acryloyl solution into a culture dish which is treated by the low-temperature plasma pretreatment step for oscillation, then carrying out ultraviolet irradiation under the conditions of introducing nitrogen and discharging oxygen, and then cleaning and drying to obtain the thermo-sensitive culture dish. The culture dish prepared by the method has a large contact angle change value, so that the culture dish is sensitive to temperature change, and the hydrophobicity and the hydrophilicity of the culture dish obviously change along with the temperature.
Description
Technical Field
The invention relates to the technical field of culture dish preparation, in particular to a preparation method of a temperature-sensitive culture dish.
Background
Poly-N-isopropylacrylamide shows obvious temperature sensitivity because macromolecules simultaneously have hydrophilic amide groups and hydrophobic isopropyl groups, when the temperature is changed at about 32 ℃, the solution and gel of the poly-N-isopropylacrylamide have reversible sudden changes with the characteristics which are just similar to the temperature of human cell culture, which provides possibility for the poly-N-isopropylacrylamide to be used for preparing temperature-sensitive culture dishes, but the mechanical strength of the poly-N-isopropylacrylamide is poor, and the poly-N-isopropylacrylamide is difficult to support to be stably formed under the condition of being swelled by water, so that the application of the poly-N-isopropylacrylamide in preparing culture dishes is limited.
On the basis, researchers try to prepare a temperature-sensitive culture dish by grafting poly-N-isopropylacrylamide (PNIPAm) on a culture dish with a preparation material with strong mechanical strength and good mechanical property through various methods. The main mechanism of PNIPAm grafting is free radical polymerization, i.e., the reaction mechanism is chain initiation, chain propagation, 3-motif reaction of chain termination, and chain transfer as a side reaction. The grafting method is generally classified into solution radical grafting, photo-initiated grafting, radiation grafting, low-temperature plasma grafting, and the like according to the graft polymerization mechanism thereof. The preparation process of the low-temperature plasma grafting method belongs to a dry processing process, does not cause pollution, is simple to operate, only modifies the surface of a high polymer material, does not influence the structure and the performance of a body, and is a relatively suitable grafting technology.
However, the inventor finds in research that the culture dish prepared by grafting the poly-N-isopropylacrylamide into the culture dish by the existing method is insensitive to temperature change, and is characterized in that the change value of the contact angle is small, and the hydrophobic property and the hydrophilic property of the culture dish are not obviously changed along with the temperature. Therefore, the preparation method of the temperature-sensitive culture dish which is sensitive to temperature change and has a large contact angle change value is provided, and the preparation method has important application value.
Disclosure of Invention
In order to solve at least one technical problem pointed out in the prior art, the invention provides a preparation method of a temperature-sensitive culture dish. The culture dish prepared by the method has a large contact angle change value, so that the culture dish is sensitive to temperature change, and the hydrophobic property and the hydrophilic property of the culture dish obviously change along with the temperature.
The technical problem to be solved by the invention is realized by the following technical scheme:
a preparation method of a temperature-sensitive culture dish comprises the following steps:
preparing a poly-N isopropyl acrylamide solution: preparing poly-N-isopropyl acrylamide solution by using water;
a culture dish pretreatment step: cleaning a culture dish, removing impurities, and drying to obtain a pretreated culture dish;
a low-temperature plasma pretreatment step: placing the pretreated culture dish in a plasma treatment device for low-temperature plasma pretreatment; taking out and exposing the pretreated product to the atmospheric environment after low-temperature plasma pretreatment;
ultraviolet irradiation grafting step: placing the poly-N isopropyl acrylamide solution into a culture dish which is treated by the low-temperature plasma pretreatment step for oscillation, then carrying out ultraviolet irradiation under the conditions of introducing nitrogen and discharging oxygen, and then cleaning and drying to obtain the thermo-sensitive culture dish.
Preferably, in the step of preparing the poly-N-isopropylacrylamide solution, the poly-N-isopropylacrylamide is taken and water is used for preparing the poly-N-isopropylacrylamide solution with the mass fraction of 32-38%.
Preferably, in the step of preparing the poly-N-isopropylacrylamide solution, the poly-N-isopropylacrylamide is taken and is prepared into the poly-N-isopropylacrylamide solution with the mass fraction of 32% by using water.
Preferably, in the step of pretreating the culture dish, the culture dish is made of polystyrene.
Preferably, the step of cleaning and impurity removing in the pretreatment step of the culture dish refers to cleaning and impurity removing by using absolute ethyl alcohol.
Preferably, the time of the low-temperature plasma pretreatment in the low-temperature plasma pretreatment step is 2 min.
Preferably, in the low-temperature plasma pretreatment step, the substrate is taken out and exposed to the atmospheric environment for 2-3 hours after low-temperature plasma pretreatment.
Preferably, in the ultraviolet irradiation grafting step, the time of ultraviolet irradiation is 15 min.
Preferably, in the step of ultraviolet irradiation grafting, the shaking refers to shaking for 10-20 min.
Preferably, in the step of ultraviolet irradiation grafting, the cleaning is carried out for 16-30 h by oscillating and cleaning in a constant-temperature water bath at 25-35 ℃.
The inventors have found in experiments how to graft poly-N-isopropylacrylamide onto a polystyrene culture dish so that it has a large contact angle variation value, which is one of the difficulties encountered by those skilled in the art. The inventor surprisingly finds out in a large number of experiments that in a plurality of reaction conditions, three factors, namely the concentration of the poly-N isopropyl acrylamide solution, the low-temperature plasma pretreatment time, the ultraviolet light irradiation time and the like, play a decisive role in determining whether the culture dish grafted with the polystyrene material of the poly-N isopropyl acryloyl has a larger contact angle change value; the culture dish of the polystyrene material grafted with the poly-N isopropyl acrylamide is prepared by the difference of any one of three factors, namely the concentration of the poly-N isopropyl acrylamide solution, the low-temperature plasma pretreatment time, the ultraviolet irradiation time and the like, and the difference of the contact angle change values is great. Only when the mass fraction of the poly-N isopropyl acryloyl solution is 32-38%, the low-temperature plasma pretreatment time is 2min, and the ultraviolet irradiation time is 15min, the prepared culture dish has a large contact angle change value; the contact angle change value of the culture dish is far higher than that of the culture dish prepared under the conditions of other poly-N isopropyl acrylamide solution concentration, low-temperature plasma pretreatment time and ultraviolet light irradiation time.
Has the advantages that: the invention provides a brand-new culture dish preparation method; the culture dish prepared by the method has a large contact angle change value, so that the culture dish is sensitive to temperature change, and the hydrophobicity and the hydrophilicity of the culture dish obviously change along with the temperature; the culture dish has wide application prospect in the field of in-vitro tissue and organ regeneration based on cell layer engineering. In addition, it can be observed through cytotoxicity experiments and cell layer detachment experiments that the cells cultured in the culture dish have good shapes and are normally attached to the wall, grow and propagate, a complete cell layer structure can be obtained by reducing the temperature after a continuous cell layer is formed, and the cells have no cytotoxicity compared with the cytotoxicity grading standard.
Detailed Description
The present invention will be further explained with reference to specific examples, which are not intended to limit the present invention in any way.
Preparation method of temperature-sensitive culture dish
(1) Preparing a poly-N isopropyl acrylamide solution: preparing poly-N-isopropyl acrylamide solution from poly-N-isopropyl acrylamide by using water;
(2) a culture dish pretreatment step: soaking and cleaning a culture dish (a polystyrene culture dish) with absolute ethyl alcohol to remove organic matters and impurities, and then drying in a drying oven in vacuum to obtain a pretreated culture dish;
(3) a low-temperature plasma pretreatment step: placing the pretreated culture dish in a plasma treatment device for low-temperature plasma pretreatment (plasma treatment in low-temperature ammonia gas, the voltage discharge frequency is 15MHz, and the power is 120W); after low-temperature plasma pretreatment, taking out and exposing the product in the atmospheric environment for 2h to obtain a peroxy group;
(4) ultraviolet irradiation grafting step: placing 2ml of poly-N-isopropylacrylamide solution into the culture dish treated by the low-temperature plasma pretreatment step, oscillating for 15min, then irradiating by ultraviolet light under the conditions of introducing nitrogen and discharging oxygen, oscillating and cleaning in a constant-temperature water bath at 30 ℃ for 24h, and placing in a drying oven for vacuum drying after cleaning to obtain the temperature-sensitive culture dish.
The differences of each embodiment and the comparative example are in the concentration of poly-N-isopropylacrylamide solution, the low-temperature plasma pretreatment time and the ultraviolet irradiation time; the contact angle change values of the culture dishes prepared in the examples and comparative examples at 37 ℃ and 20 ℃ are tested simultaneously; see table 1 for details.
Table 1.
As can be seen from the experimental data in Table 1, the petri dish prepared in example 1 has a large contact angle variation value. In particular, the petri dish prepared in example 1 had a contact angle variation value as high as 24.3 °. According to tests, the contact angle of the bottom wall surface of the culture dish prepared in example 1 at 37 ℃ is 80.4 degrees, the culture dish is relatively hydrophobic, the contact angle at 20 ℃ is 56.1 degrees, the culture dish is relatively hydrophilic, and obvious wettability change of the culture dish grafted with poly-N isopropyl acryloyl along with temperature change can be seen.
As can be seen from the experimental data in Table 1, the difference of the contact angle change values of the culture dishes prepared in comparative examples 1-16 is huge and is far smaller than that of example 1. This indicates that: the concentration of poly-N isopropyl acryloyl solution, the low-temperature plasma pretreatment time, the ultraviolet light irradiation time and other three factors play a decisive role in determining whether a culture dish grafted with poly-N isopropyl acryloyl polystyrene material has a larger contact angle change value; the culture dish of the polystyrene material grafted with the poly-N isopropyl acryloyl is prepared by the difference of any one of three factors, namely the concentration of the poly-N isopropyl acryloyl solution, the low-temperature plasma pretreatment time, the ultraviolet light irradiation time and the like, and the difference of the contact angle change values of the culture dish is huge. Only when the mass fraction of the poly-N isopropyl acryloyl solution is 32-38%, the low-temperature plasma pretreatment time is 2min, and the ultraviolet irradiation time is 15min, the prepared culture dish has a large contact angle change value; the contact angle change value of the culture dish is far higher than that of the culture dish prepared under the conditions of other poly-N isopropyl acryloyl solution concentration, low-temperature plasma pretreatment time and ultraviolet light irradiation time.
Claims (10)
1. A preparation method of a temperature-sensitive culture dish is characterized by comprising the following steps:
preparing a poly-N isopropyl acrylamide solution: preparing poly-N-isopropyl acrylamide solution from poly-N-isopropyl acrylamide by using water;
a culture dish pretreatment step: cleaning a culture dish, removing impurities, and drying to obtain a pretreated culture dish;
a low-temperature plasma pretreatment step: placing the pretreated culture dish in a plasma treatment device for low-temperature plasma pretreatment; taking out the pretreated substrate after low-temperature plasma pretreatment and exposing the pretreated substrate to the atmospheric environment;
ultraviolet irradiation grafting step: placing the poly-N isopropyl acrylamide solution into a culture dish treated by the low-temperature plasma pretreatment step for oscillation, then carrying out ultraviolet irradiation under the conditions of introducing nitrogen and discharging oxygen, and then cleaning and drying to obtain the thermo-sensitive culture dish.
2. The method for preparing a temperature-sensitive culture dish according to claim 1, wherein in the step of preparing the poly-N-isopropylacrylamide solution, the poly-N-isopropylacrylamide is taken and water is used for preparing the poly-N-isopropylacrylamide solution with the mass fraction of 32-38%.
3. The method for preparing a temperature-sensitive culture dish according to claim 2, wherein in the step of preparing the poly-N-isopropylacrylamide solution, the poly-N-isopropylacrylamide is taken and is prepared into the poly-N-isopropylacrylamide solution with the mass fraction of 32% by using water.
4. The method of preparing a temperature-sensitive culture dish according to claim 1, wherein the culture dish is a polystyrene culture dish in the step of pretreating the culture dish.
5. The method for preparing a temperature-sensitive culture dish according to claim 1, wherein the step of washing and removing impurities in the pretreatment step of the culture dish is washing and removing impurities with absolute ethyl alcohol.
6. The method for preparing a temperature-sensitive culture dish according to claim 1, wherein the time for the low-temperature plasma pretreatment in the low-temperature plasma pretreatment step is 2 min.
7. The method for preparing a temperature-sensitive culture dish according to claim 1, wherein in the low-temperature plasma pretreatment step, the culture dish is taken out and exposed to the atmosphere for 2-3 hours after the low-temperature plasma pretreatment.
8. The method of manufacturing a temperature-sensitive culture dish according to claim 1, wherein in the ultraviolet irradiation grafting step, the time of ultraviolet irradiation is 15 min.
9. The method for preparing a temperature-sensitive culture dish according to claim 1, wherein in the step of ultraviolet irradiation grafting, the shaking is shaking for 10-20 min.
10. The method for preparing a temperature-sensitive culture dish according to claim 1, wherein in the step of ultraviolet irradiation grafting, the washing is carried out in a constant temperature water bath at 25 to 35 ℃ for 16 to 30 hours under shaking.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210575187.2A CN114920972A (en) | 2022-05-25 | 2022-05-25 | Preparation method of temperature-sensitive culture dish |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210575187.2A CN114920972A (en) | 2022-05-25 | 2022-05-25 | Preparation method of temperature-sensitive culture dish |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11349643A (en) * | 1998-06-05 | 1999-12-21 | Nitta Gelatin Inc | Temperature-sensitive polymeric compound, its production, temperature-sensitive polymer composition, and cell culture substrate |
| CN101565489A (en) * | 2009-06-02 | 2009-10-28 | 中山大学 | Preparation method of polystyrene with thermo-sensitive surface |
| CN102276866A (en) * | 2011-07-18 | 2011-12-14 | 海狸（广州）生物科技有限公司 | Plasma-photochemical method for grafting carboxyl on cell culture surface |
| CN110938323A (en) * | 2018-09-25 | 2020-03-31 | 广州洁特生物过滤股份有限公司 | Temperature-sensitive cell culture surface and preparation method thereof |
| CN113265032A (en) * | 2021-04-30 | 2021-08-17 | 华南师范大学 | Preparation method and application of polyacrylamide modified temperature-sensitive copolymer |
-
2022
- 2022-05-25 CN CN202210575187.2A patent/CN114920972A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11349643A (en) * | 1998-06-05 | 1999-12-21 | Nitta Gelatin Inc | Temperature-sensitive polymeric compound, its production, temperature-sensitive polymer composition, and cell culture substrate |
| CN101565489A (en) * | 2009-06-02 | 2009-10-28 | 中山大学 | Preparation method of polystyrene with thermo-sensitive surface |
| CN102276866A (en) * | 2011-07-18 | 2011-12-14 | 海狸（广州）生物科技有限公司 | Plasma-photochemical method for grafting carboxyl on cell culture surface |
| CN110938323A (en) * | 2018-09-25 | 2020-03-31 | 广州洁特生物过滤股份有限公司 | Temperature-sensitive cell culture surface and preparation method thereof |
| CN113265032A (en) * | 2021-04-30 | 2021-08-17 | 华南师范大学 | Preparation method and application of polyacrylamide modified temperature-sensitive copolymer |
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