A low surface free energy culture dish and a method for preparing the same

Abstract

The present application relates to the technical field of experimental consumables, in particular to a low surface free energy culture dish and a preparation method thereof. The present application discloses a low surface free energy culture dish and a preparation method thereof. The material of the culture dish comprises: 75-80 parts by weight of polystyrene particles; 10-40 parts by weight of a polymer; 1-5 parts by weight of a plasticizer; and 1-5 parts by weight of an anti-aging agent, wherein the polymer comprises a high polymer and a block copolymer. The culture dish provided by the present application has high light transmittance, is resistant to scratching, has aging resistance, and is suitable for wide promotion of stem cell research. The preparation method of the culture dish provided by the present application simplifies the process flow, directly uses an extrusion process, and reduces the emission of inhalable toxic and harmful substances compared with a vacuum coating process.

Description

Technical Field

[0001] This invention relates to the field of laboratory consumables technology, specifically to a low surface free energy petri dish and its preparation method. Background Technology

[0002] A petri dish is a laboratory vessel used for culturing microorganisms or cells. It consists of a flat, disc-shaped base and a lid, and is typically made of glass or plastic. Petri dishes can be used for the culture of plant materials, microorganisms, and adherent cultures of animal cells. Plastic petri dishes are mostly made of PS (polystyrene). These types of dishes, due to their high surface free energy at the bottom, are not conducive to cell membrane detachment.

[0003] Cell membranes refer to complete monolayer cell membranes obtained by culturing isolated cells on specific materials under in vitro culture conditions and separating the cells from the culture material without damaging cell connections. Compared with cell injection and polymer-cell chimeric implants, cell membranes have the following advantages in clinical applications: (1) After traditional intravenous / arterial cell injection, the distribution tends to be biased towards tissues such as the liver and lungs, making it difficult to target mesenchymal stem cells to the transplant organ site; while cell membranes can be directly transplanted to the required site during transplantation surgery; (2) Traditional cell system injections are often difficult to achieve good therapeutic effects for diseases in certain special sites, such as bones, joints, and tendons, due to the lack of rich capillary networks in the tissues; while cell membranes can be directly applied to these sites and play a key therapeutic role; (3) Cell membranes do not require the support of biodegradable polymer materials, so they are less likely to trigger immune and inflammatory reactions caused by polymer materials; (4) Cell membranes completely preserve the extracellular matrix, which is conducive to cell regeneration and in vivo utilization.

[0004] To ensure the integrity of the prepared cell membrane, one method involves culturing a monolayer of cells in a low surface energy culture dish and then detaching the cell membrane. However, the preparation of such low surface energy culture dishes often employs vacuum coating technology, which is costly and involves the emission of some inhalable toxic and harmful substances.

[0005] Therefore, there is an urgent need to develop a method for preparing low surface energy petri dishes that has a simple process and reduces the emission of harmful substances. Summary of the Invention

[0006] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one object of this invention is to provide a low surface free energy petri dish and a method for preparing the same.

[0007] Therefore, in a first aspect, the present invention provides a petri dish. According to an embodiment of the present invention, the material of the petri dish comprises:

[0008] (1) 75-80 parts by weight of polystyrene granules;

[0009] (2) 10-40 parts by weight of polymer;

[0010] (3) 1-5 parts by weight of plasticizer;

[0011] (4) 1-5 parts by weight of anti-aging agent,

[0012] The polymer comprises a high polymer and a block copolymer, wherein the monomers constituting the high polymer or the block copolymer are selected from at least one of the following:

[0013] Vinylimidazole, vinylpyrrolidone, aminostyrene, methacrylamide, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, N,N-dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride chloride), 2-vinylpyridine, acrylonitrile, hexalactone, N,N-Dimethylaminomethyl styrene, 4-vinylpyridine, divinylbenzene, vinyl benzate, benzyl methacrylate, cyclohexylmethacrylate, butyl methacrylate, isopropyl methacrylate, acrylamide, allyl methacrylate, 2-isocyanatoethyl methacrylate, ethylene glycol dimethacrylate, di(ethylene glycol)methyl ester methacrylate, hydroxyethyl methacrylate, 1,2,4-trivinylcyclohexane, furfuryl methacrylate, tetrahydrofurfuryl methacrylate furfuryl methacrylate), hexylmethacrylate, hydroxyethyl methacrylate, glycidyl methacrylate, propargyl methacrylate, 1,4-butanediol divinyl ether, isobornyl acrylate, ethylene glycol diacrylate, propargyl acrylate, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane, hexavinyldisiloxane, 2,4,6-trivinyl-2,4,6-trimethylcyclotrisiloxane3,5-trimethylcyclotrisiloxane, 2,4,6-trimethyl-2,4,6-trivinylcyclotrisilazane, dimethylphenylvinylsilane, heptadecafluorodecyl methacrylate, perfluorodecyl acrylate, heptafluorobutylmethacrylate, 1,1,1,3,3,3-hexafluoroisopropyl methacrylate, 2,2,3,3,4,4-hexafluoro-1,5-pentyl diacrylate, 2-(perfluorohexyl)ethyl methacrylate methacrylate), 2,2,2-trifluoroethylmethacrylate, pentafluorophenyl methacrylate, 1H,1H,7H-dodecafluoroheptyl acrylate, 1H,1H,2H,2H-heptadecafluorodecyl acrylate, diethylene glycol divinyl ether, 1,9-decadiene, 2-methacrylic anhydride, 1,2,4-trivinylcyclohexane, allyl acetoacetate, maleic anhydride anhydride), 4-vinylaniline, 9-vinylcarbazole, 2-dimethylaminoethyl acrylate, N-acrylateThe following are listed: N-diethylaminoethyl acrylate, 2-(dimethylamino)ethyl acrylate, 3-(dimethylamino)propyl acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(tert-butylamino)ethyl methacrylate, N,N-dimethylaminomethylstyrene, methacrylic acid, acrylamide, vinyl-N-methylpyridinium chloride, and N,N-(4-vinylbenzyl)-N,N-dimethylamine.

[0014] The inventors discovered that when culture dishes prepared using the aforementioned special raw materials are used to culture cells, such as stem cells, especially when cultured in liquid culture medium, the cells can adhere to the bottom of the culture space during the expansion process and proliferate by adhering to the bottom of the culture dish. Furthermore, the surface free energy at the bottom of the culture dish does not exceed 90 mJ / m². 2 By adjusting the ion concentration in the cell culture medium, the binding force between the culture dish and the cells can be reduced, allowing adherent cells to detach from the bottom of the culture dish as a cell membrane, thus effectively obtaining a cell membrane. The culture dish provided by this invention, due to its low surface free energy, allows for the reduction of the binding force between the culture dish and the cells by adjusting the ion concentration in the cell culture medium, thereby enabling adherent cells to detach from the bottom of the culture dish as a cell membrane, effectively obtaining a cell membrane.

[0015] According to an embodiment of the present invention, the petri dish may further have at least one of the following additional technical features:

[0016] According to an embodiment of the invention, the polymer comprises a butyl methacrylate polymer and a poly(2-vinylpyridine-co-styrene) block copolymer. This results in better detachment of the cell membrane from the culture dish.

[0017] According to an embodiment of the present invention, the weight ratio of the butyl methacrylate polymer to the poly(2-vinylpyridine-co-styrene) block copolymer is 100:(15-30). Maintaining this weight ratio within this range allows for a smoother surface in the culture dish, maintains a lower surface free energy at the bottom of the dish, facilitates cell membrane detachment, and ensures the integrity of the cell membrane.

[0018] According to an embodiment of the present invention, the plasticizer is selected from at least one of diisooctyl phthalate, neopentyl glycol dibenzoate, diisooctyl cyclohexane-1,2-dicarboxylate, diisononyl cyclohexane-1,2-dicarboxylate, and tributyl acetylacetate.

[0019] According to an embodiment of the present invention, the anti-aging agent is selected from at least one of pentaerythritol tetrakis[β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate] (antioxidant 1010), octadecyl β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate (antioxidant 1076), dilauryl thiodipropionate (DLTP), and distearate thiodipropionate (DSTP). These specific types of anti-aging agents can further improve the scratch resistance and aging resistance of the petri dish.

[0020] According to an embodiment of the present invention, the surface free energy of the petri dish does not exceed 90 mJ / m 2 Therefore, the cell membrane detaches more effectively from the culture dish.

[0021] According to an embodiment of the present invention, the surface free energy of the petri dish does not exceed 60 mJ / m 2 .

[0022] A second aspect of the present invention provides a method for preparing a petri dish. According to an embodiment of the present invention, the petri dish is pressed from the following raw materials:

[0023] (1) 75-80 parts by weight of polystyrene granules;

[0024] (2) 10-40 parts by weight of polymer;

[0025] (3) 1-5 parts by weight of plasticizer;

[0026] (4) 1-5 parts by weight of anti-aging agent,

[0027] The polymer includes high polymers and block copolymers.

[0028] The monomers constituting the polymer or block copolymer are selected from at least one of the following:

[0029] Vinylimidazolium, vinylpyrrolidone, aminostyrene, methacrylamide, dimethacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, cyanomethylstyrene, 1-methylpyridine chloride, vinylcaprolactam, acrylic acid, N,N-dimethylaminoethyl acrylate, ethyl chloride acrylate, cyanoethyl acrylate, propyl 3-(dimethylamino)acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, caprolactone, N,N-dimethylvinylbenzeneamine, 4-vinylpyridine, divinylbenzene, vinyl benzoate, benzyl methacrylate, cyclohexyl methacrylate, butyl methacrylate, methyl Isopropyl acrylate, Acrylamide, Allyl methacrylate, Isocyanoethyl methacrylate, Ethylene glycol dimethacrylate, Ethylene glycol methyl methacrylate, Polyhydroxyethyl methacrylate, 1,2,4-Triethylcyclohexane, Methyl furfuryl alcohol, Tetrahydrofurfuryl methacrylate, Hexyl methacrylate, Hydroxyethyl methacrylate, Glycidyl methacrylate, Propylene methacrylate, 1,4-Butanediol vinyl ether, Isoborneol acrylate, Ethylene glycol diacrylate, Propylene acrylate, 2,4,6,8-Tetramethyl-2,4,6,8-Tetravinylcyclotetrasiloxane, Hexavinyldisiloxane, 2,4 6-Trivinyl-2,4,6-trimethylcyclotrisiloxane, trivinyltrimethylcyclotrisilazane, dimethylphenylvinylsilane, (perfluorooctyl)ethyl methacrylate, perfluorodecyl acrylate, heptafluorobutyl methacrylate, 1,1,1,3,3,3-hexafluoroisopropyl isobutylene acrylate, 2,2,3,3,4,4-hexafluoro-1,5-pentamethylsilyl acrylate, 2-(perfluorohexyl)ethyl methacrylate, trifluoroethyl methacrylate, pentafluorophenyl methacrylate, 1H,1H,7H-dodecylheptyl acrylate, 1H,1H,2H,2H-perfluorodecyl acrylate Diethylene glycol divinyl ether, 1,9-decadiene, 2-methacrylic anhydride, 1,2,4-trivinylcyclohexane, allyl acetoacetate, maleic anhydride, 4-vinylaniline, 9-vinylcarbazole, dimethylaminoethyl acrylate, N,N-diethylaminoethyl acrylate, 2-(dimethylamino)acrylate, 3-(dimethylamino)acrylate, 2-(dimethylamino)methacrylate, 2-(tert-butylamino)methacrylate, N,N-dimethylvinylanisole, methacrylic acid, acrylamide, vinyl-N-methylpyridine chloride, N,N-dimethylvinylanisole.

[0030] The inventors discovered that culture dishes prepared using this method have low surface free energy, enabling rapid detachment of cell membranes from the bottom of the culture dish. This is likely because changing the ion concentration in the culture medium, such as calcium and / or magnesium ions, alters the conformation of the transmembrane receptors of the focal adhesion complex, reducing the binding force between the culture dish bottom and the cells. This allows cells to detach from the bottom surface of the culture dish without trypsin hydrolysis of intercellular proteins, resulting in intact cell membranes. The culture dishes prepared by this invention, due to their low surface free energy, allow for adjustments to the ion concentration in the cell culture medium, reducing the binding force between the culture dish and cells. This enables adherent cells to detach from the bottom of the culture dish as cell membranes, effectively obtaining the cell membrane. The culture dishes prepared by this method have high light transmittance, are scratch-resistant, and possess aging resistance, making them suitable for widespread application in stem cell research.

[0031] Traditional polystyrene culture dishes have high surface free energy, allowing cells to only adhere to the surface for growth. This invention adds specific proportions of the aforementioned polymers and block copolymers to the polystyrene, thereby reducing the surface free energy of the mixed material. Consequently, cells cultured in dishes made from this material exhibit reduced adhesion, allowing them to be manually detached for biomedical applications.

[0032] The method for preparing culture dishes provided by this invention simplifies the process flow by directly using an extrusion process, which reduces the emission of inhalable toxic and harmful substances compared to the vacuum coating process. At the same time, the culture dishes prepared by this method have a smooth surface, maintain a low surface free energy at the bottom of the culture dish, and allow the cell membrane to detach successfully while ensuring the integrity of the cell membrane.

[0033] According to an embodiment of the invention, the polymer comprises a butyl methacrylate polymer and a poly(2-vinylpyridine-co-styrene) block copolymer. This results in better detachment of the cell membrane from the culture dish.

[0034] According to an embodiment of the present invention, the weight ratio of the butyl methacrylate polymer to the poly(2-vinylpyridine-co-styrene) block copolymer is 100:(15-30). Maintaining this weight ratio within this range allows for a smoother surface in the culture dish, maintains a lower surface free energy at the bottom of the dish, facilitates cell membrane detachment, and ensures the integrity of the cell membrane.

[0035] According to an embodiment of the present invention, the plasticizer is selected from at least one of diisooctyl phthalate, neopentyl glycol dibenzoate, diisooctyl cyclohexane-1,2-dicarboxylate, diisononyl cyclohexane-1,2-dicarboxylate, and tributyl acetylacetate.

[0036] According to an embodiment of the present invention, the anti-aging agent is selected from at least one of pentaerythritol tetrakis[β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate] (antioxidant 1010), octadecyl β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate (antioxidant 1076), dilauryl thiodipropionate (DLTP), and distearate thiodipropionate (DSTP).

[0037] According to an embodiment of the present invention, the pressing process includes:

[0038] (A) The polystyrene particles and polymer are mixed and heated at 150-160°C for 10-12 hours to obtain the first mixture M1;

[0039] (B) Add the plasticizer and anti-aging agent to the first mixture M1, knead, and obtain the second mixture M2;

[0040] (C) The second mixture M2 is injection molded to obtain the culture dish.

[0041] The culture dish prepared by the method of the present invention has a bottom surface free energy of no more than 90 mJ / m². 2 Therefore, by adjusting the ion concentration in the cell culture medium, the binding force between the cell and the bottom of the culture dish can be reduced, allowing adherent cells to detach from the bottom of the dish as a cell membrane, effectively obtaining an intact cell membrane. This method simplifies the process by directly employing extrusion, reducing the emission of inhalable toxic substances compared to vacuum coating. Furthermore, the culture dish prepared by this method has a smooth surface, maintaining a low surface free energy at the bottom, enabling successful cell membrane detachment while ensuring the integrity of the cell membrane.

[0042] According to an embodiment of the present invention, the pressing process further includes:

[0043] Before proceeding to step (B), the first mixture M1 is cooled to 20-30°C.

[0044] According to an embodiment of the present invention, the cooling rate is no greater than 1.0℃ / min.

[0045] The method for preparing culture dishes provided by this invention employs a specific cooling rate (not greater than 1.0℃ / min) before step (B) to allow the polymer sol to recover from fatigue during slow cooling, relax the mechanical stress experienced during the initial mixing, and reduce shrinkage of the rubber compound. Simultaneously, during the slow cooling and resting process, polymers of different components can continue to diffuse and react in the rubber compound, enhancing the uniformity of material dispersion. If the cooling rate of the rubber compound is too fast, it will affect the material properties, leading to warping and deformation of the subsequent finished product.

[0046] According to an embodiment of the present invention, in step (B), the kneading speed is 400-600 rpm and the time is 5-15 min.

[0047] According to an embodiment of the present invention, in step (C), the injection molding process includes:

[0048] (a) The second mixture M2 is fed into an injection molding machine and extruded into a hot melt state, wherein the barrel temperature of the injection molding machine is 200-250°C;

[0049] (b) The hot-molten melt is injected into the mold and held under pressure for 2-4 seconds to obtain the culture dish.

[0050] The third aspect of this invention provides the use of the culture dish described in the first aspect or the culture dish prepared by the preparation method described in the second aspect in the preparation of cell films.

[0051] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0052] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0053] Figure 1 This is a schematic diagram of a petri dish according to an embodiment of the present invention, wherein 100: petri dish body; 110: culture space; 200: petri dish base plate;

[0054] Figure 2 This is a schematic diagram of the structure of a PVDF membrane according to an embodiment of the present invention, 300: PVDF membrane;

[0055] Figure 3 This is a schematic diagram of the structure of a PVDF membrane according to another embodiment of the present invention, 300: PVDF membrane;

[0056] Figure 4This is a schematic diagram of the structure of the petri dish prepared by the method in Example 1. 1: Petri dish body; 2: Antibacterial cap; 3: Petri dish base plate;

[0057] Figure 5 This is a schematic diagram illustrating the principle of cell membrane separation from the culture dish in Example 2;

[0058] Figure 6 The figures show the state of cell membranes obtained by culturing mesenchymal stem cells in a commercially available styrene culture dish in Example 3 and a culture dish prepared by the method of the present invention in Example 1, as well as the state of the cell membranes after being peeled off from the bottom of the culture dish. Figure A shows the cell membrane obtained by culturing mesenchymal stem cells in a commercially available styrene culture dish; Figure B shows the state of the cell membrane after being peeled off from the bottom of the culture dish in Figure A; Figure C shows the cell membrane obtained by culturing mesenchymal stem cells in a culture dish using the culture dish of Example 1; and Figure D shows the state of the cell membrane after being peeled off from the bottom of the culture dish in Figure C. Detailed Implementation

[0059] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0060] In the description of this invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0061] Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," etc., may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0062] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0063] petri dish

[0064] According to a specific embodiment of the present invention, the present invention provides a petri dish, the material of which includes:

[0065] (1) 75-80 parts by weight of polystyrene granules;

[0066] (2) 10-40 parts by weight of polymer;

[0067] (3) 1-5 parts by weight of plasticizer;

[0068] (4) 1-5 parts by weight of anti-aging agent,

[0069] The polymer comprises a high polymer and a block copolymer, wherein the monomers constituting the high polymer or the block copolymer are selected from at least one of the following:

[0070] Vinylimidazolium, vinylpyrrolidone, aminostyrene, methacrylamide, dimethacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, cyanomethylstyrene, 1-methylpyridine chloride, vinylcaprolactam, acrylic acid, N,N-dimethylaminoethyl acrylate, ethyl chloride acrylate, cyanoethyl acrylate, propyl 3-(dimethylamino)acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, caprolactone, N,N-dimethylvinylbenzeneamine, 4-vinylpyridine, divinylbenzene, vinyl benzoate, benzyl methacrylate, cyclohexyl methacrylate, butyl methacrylate, methyl Isopropyl acrylate, Acrylamide, Allyl methacrylate, Isocyanoethyl methacrylate, Ethylene glycol dimethacrylate, Ethylene glycol methyl methacrylate, Polyhydroxyethyl methacrylate, 1,2,4-Triethylcyclohexane, Methyl furfuryl alcohol, Tetrahydrofurfuryl methacrylate, Hexyl methacrylate, Hydroxyethyl methacrylate, Glycidyl methacrylate, Propylene methacrylate, 1,4-Butanediol vinyl ether, Isoborneol acrylate, Ethylene glycol diacrylate, Propylene acrylate, 2,4,6,8-Tetramethyl-2,4,6,8-Tetravinylcyclotetrasiloxane, Hexavinyldisiloxane, 2,4 6-Trivinyl-2,4,6-trimethylcyclotrisiloxane, trivinyltrimethylcyclotrisilazane, dimethylphenylvinylsilane, (perfluorooctyl)ethyl methacrylate, perfluorodecyl acrylate, heptafluorobutyl methacrylate, 1,1,1,3,3,3-hexafluoroisopropyl isobutylene acrylate, 2,2,3,3,4,4-hexafluoro-1,5-pentamethylsilyl acrylate, 2-(perfluorohexyl)ethyl methacrylate, trifluoroethyl methacrylate, pentafluorophenyl methacrylate, 1H,1H,7H-dodecylheptyl acrylate, 1H,1H,2H,2H-perfluorodecyl acrylate Diethylene glycol divinyl ether, 1,9-decadiene, 2-methacrylic anhydride, 1,2,4-trivinylcyclohexane, allyl acetoacetate, maleic anhydride, 4-vinylaniline, 9-vinylcarbazole, dimethylaminoethyl acrylate, N,N-diethylaminoethyl acrylate, 2-(dimethylamino)acrylate, 3-(dimethylamino)acrylate, 2-(dimethylamino)methacrylate, 2-(tert-butylamino)methacrylate, N,N-dimethylvinylanisole, methacrylic acid, acrylamide, vinyl-N-methylpyridine chloride, N,N-dimethylvinylanisole.

[0071] According to a specific embodiment of the present invention, the polymer comprises a high polymer and a block copolymer. The monomers constituting the high polymer can be any one or more of the aforementioned monomer set, or the high polymer can be a mixture of multiple high polymers. The monomers constituting the block copolymer can be any two or more of the aforementioned monomer set, or the block copolymer can be a mixture of multiple block copolymers. For example, the material of the culture dish comprises a butyl methacrylate polymer and a poly(2-vinylpyridine-co-styrene) block copolymer, wherein the weight ratio of the butyl methacrylate polymer to the poly(2-vinylpyridine-co-styrene) block copolymer is 100:(15-30).

[0072] According to a specific embodiment of the present invention, the plasticizer is selected from at least one of diisooctyl phthalate, neopentyl glycol dibenzoate, diisooctyl cyclohexane-1,2-dicarboxylate, diisononyl cyclohexane-1,2-dicarboxylate, and tributyl acetylacetonate; the anti-aging agent is selected from at least one of pentaerythritol tetrakis[β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate] (antioxidant 1010), octadecyl β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate (antioxidant 1076), dilauryl thiodipropionate (DLTP), and distearate thiodipropionate (DSTP).

[0073] It should be noted that the plasticizers and anti-aging agents contained in the petri dish material provided by the present invention are not limited to the above-mentioned types, but may also be various other plasticizers and anti-aging agents known in the art and used in the injection molding field, all of which are included within the protection scope of the present invention.

[0074] According to a specific embodiment of the present invention, the present invention provides a petri dish, with reference to... Figure 1 The culture dish body 100 defines a culture space 110 and a culture dish base plate 200. The surface free energy of the culture dish base plate 200 does not exceed 90 mJ / m². 2 More preferably, the surface free energy of the petri dish base 200 does not exceed 60 mJ / m². 2 Therefore, the cell membrane detaches more effectively from the culture dish.

[0075] The specific shape of the culture dish body 100 is not particularly limited, and those skilled in the art can choose according to actual needs. According to some embodiments of the present invention, the overall shape of the culture dish is circular, its cell-bearing surface is smooth and flat, its height is 13–17 cm, its diameter is 35–100 mm, and its effective cell culture area is 8–57 cm². 2 .

[0076] According to one embodiment of the present invention, the culture dish body 100 is made of polystyrene, which has good light transmittance and facilitates observation of cell morphology. In some embodiments of the present invention, the light transmittance of the culture dish body to visible light with a wavelength of 400-800 nm is above 80%.

[0077] According to a preferred embodiment of the present invention, the polymer contained in the culture dish material includes butyl methacrylate polymer and poly(2-vinylpyridine-co-styrene) block copolymer, and the weight ratio between the two is 100:(1530). The ratio between the polymer and the block copolymer can be adjusted according to the different polymers and block copolymers contained in the polymer.

[0078] Preparation method of petri dish

[0079] According to a specific embodiment of the present invention, the present invention provides a method for preparing a petri dish, wherein the petri dish is pressed from the following raw materials:

[0080] (1) 75-80 parts by weight of polystyrene granules;

[0081] (2) 10-40 parts by weight of polymer;

[0082] (3) 1-5 parts by weight of plasticizer;

[0083] (4) 1-5 parts by weight of anti-aging agent,

[0084] The polymer includes high polymers and block copolymers.

[0085] The monomers constituting the polymer or block copolymer are selected from at least one of the following:

[0086] Vinylimidazolium, vinylpyrrolidone, aminostyrene, methacrylamide, dimethacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, cyanomethylstyrene, 1-methylpyridine chloride, vinylcaprolactam, acrylic acid, N,N-dimethylaminoethyl acrylate, ethyl chloride acrylate, cyanoethyl acrylate, propyl 3-(dimethylamino)acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, caprolactone, N,N-dimethylvinylbenzeneamine, 4-vinylpyridine, divinylbenzene, vinyl benzoate, benzyl methacrylate, cyclohexyl methacrylate, butyl methacrylate, methyl Isopropyl acrylate, Acrylamide, Allyl methacrylate, Isocyanoethyl methacrylate, Ethylene glycol dimethacrylate, Ethylene glycol methyl methacrylate, Polyhydroxyethyl methacrylate, 1,2,4-Triethylcyclohexane, Methyl furfuryl alcohol, Tetrahydrofurfuryl methacrylate, Hexyl methacrylate, Hydroxyethyl methacrylate, Glycidyl methacrylate, Propylene methacrylate, 1,4-Butanediol vinyl ether, Isoborneol acrylate, Ethylene glycol diacrylate, Propylene acrylate, 2,4,6,8-Tetramethyl-2,4,6,8-Tetravinylcyclotetrasiloxane, Hexavinyldisiloxane, 2,4 6-Trivinyl-2,4,6-trimethylcyclotrisiloxane, trivinyltrimethylcyclotrisilazane, dimethylphenylvinylsilane, (perfluorooctyl)ethyl methacrylate, perfluorodecyl acrylate, heptafluorobutyl methacrylate, 1,1,1,3,3,3-hexafluoroisopropyl isobutylene acrylate, 2,2,3,3,4,4-hexafluoro-1,5-pentamethylsilyl acrylate, 2-(perfluorohexyl)ethyl methacrylate, trifluoroethyl methacrylate, pentafluorophenyl methacrylate, 1H,1H,7H-dodecylheptyl acrylate, 1H,1H,2H,2H-perfluorodecyl acrylate The polymer comprises diethylene glycol divinyl ether, 1,9-decadiene, 2-methacrylic anhydride, 1,2,4-trivinylcyclohexane, allyl acetoacetate, maleic anhydride, 4-vinylaniline, 9-vinylcarbazole, dimethylaminoethyl acrylate, N,N-diethylaminoethyl acrylate, 2-(dimethylamino)acrylate, 3-(dimethylamino)acrylate, 2-(dimethylamino)methacrylate, 2-(tert-butylamino)methacrylate, N,N-dimethylvinylanisole, methacrylic acid, acrylamide, vinyl-N-methylpyridine chloride, and N,N-dimethylvinylanisole. Preferably, the polymer comprises a butyl methacrylate polymer and a poly(2-vinylpyridine-co-styrene) block copolymer.

[0087] According to a specific embodiment of the present invention, the plasticizer is selected from at least one of diisooctyl phthalate, neopentyl glycol dibenzoate, diisooctyl cyclohexane-1,2-dicarboxylate, diisononyl cyclohexane-1,2-dicarboxylate, and tributyl acetylacetonate; the anti-aging agent is selected from at least one of pentaerythritol tetrakis[β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate] (antioxidant 1010), octadecyl β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate (antioxidant 1076), dilauryl thiodipropionate (DLTP), and distearate thiodipropionate (DSTP). It should be noted that the plasticizers and anti-aging agents contained in the petri dish material provided by the present invention are not limited to the above-mentioned types, but may also be various other plasticizers and anti-aging agents known in the art and used in the injection molding field, all of which are included within the scope of protection of the present invention.

[0088] According to a specific embodiment of the present invention, the pressing process includes:

[0089] (A) The polystyrene particles and polymer are mixed and heated at 150-160°C for 10-12 hours to obtain the first mixture M1;

[0090] (B) Add the plasticizer and anti-aging agent to the first mixture M1, knead, and obtain the second mixture M2;

[0091] (C) The second mixture M2 is injection molded to obtain the culture dish.

[0092] According to a specific embodiment of the present invention, the pressing process further includes:

[0093] Before proceeding to step (B), the first mixture M1 is cooled to 20-30°C, and the cooling rate is no greater than 1.0°C / min.

[0094] According to a specific embodiment of the present invention, in step (B), the kneading speed is 400-600 rpm, and the time is 5-15 min. According to a specific embodiment of the present invention, in step (C), the injection molding process includes:

[0095] (a) The second mixture M2 is fed into an injection molding machine and extruded into a hot melt state, wherein the barrel temperature of the injection molding machine is 200-250°C;

[0096] (b) The hot-molten melt is injected into the mold and held under pressure for 2-4 seconds to obtain the culture dish.

[0097] According to a more specific embodiment of the present invention, the petri dish provided by the present invention is made by mixing and high-temperature pressing the following raw materials in parts by weight: A, 75-80 parts of polystyrene (PS) particles; B, 10-20 parts of butyl methacrylate polymer; C, 10-20 parts of poly(2-vinylpyridine-co-styrene) block copolymer; D, 1-5 parts of plasticizer; E, 1-5 parts of anti-aging agent. The specific preparation steps of the petri dish include:

[0098] G1. Add polystyrene particles, butyl methacrylate, and poly(2-vinylpyridine-co-styrene) block copolymer to a reaction vessel in a certain proportion, stir evenly, heat to 150-160℃, react for 10-12 hours, cool to room temperature, and obtain mixture N1.

[0099] G2. Add plasticizer and anti-aging agent to the mixture in proportion, and knead at 500 rpm ± 20% for 10 min to obtain mixture N2.

[0100] G3. The hot-melt plasticized N2 is fed into an injection molding machine preheated to 220℃. Under the action of the screw, it is pressed into a hot-melt state and injected into a petri dish mold. The pressure is held for 2-4 seconds, and the mold is opened after cooling.

[0101] In step G1, the cooling rate to room temperature is <1.0℃ / min, and in step G3, the injection pressure is 80MPa (±10%).

[0102] Applications in the preparation of cell membranes

[0103] According to a specific embodiment of the present invention, the present invention provides the use of the culture dish described above or prepared by the above method in the preparation of cell films. As mentioned above, the free energy at the bottom of the culture space of this culture medium does not exceed 90 mJ / m. 2 Therefore, by adjusting the ion concentration in the cell culture medium, the binding force between the bottom of the culture dish and the cells can be reduced, allowing adherent cells to detach from the bottom of the culture dish as a cell membrane, thus effectively obtaining the cell membrane.

[0104] Cell membrane preparation methods, composite films

[0105] According to a specific embodiment of the present invention, the present invention provides a method for preparing a cell membrane, comprising:

[0106] 1) The cells are cultured in the above-mentioned culture dishes using liquid culture medium in order to form a monolayer cell film;

[0107] 2) The monolayer cell membrane is placed in a stripping solution, wherein the cation concentration in the stripping solution is lower than that in the liquid culture medium, and the cation includes at least one of calcium ions and / or magnesium ions; and

[0108] 3) Collect the monolayer cell membrane from the stripping solution.

[0109] The method for preparing cell films according to embodiments of the present invention will be described in further detail below.

[0110] The method for preparing cell membranes proposed in this invention does not particularly limit the specific type of cells. For example, the cells can be at least one selected from stem cells, neurons, astrocytes, oligodendrocytes, epithelial cells, endothelial cells, myocytes, and fibroblasts. Specifically, stem cells include at least one selected from induced pluripotent stem cells, adult stem cells, mesenchymal stem cells, neural stem cells, cardiac stem cells, and lung stem cells; the mesenchymal stem cells include at least one selected from bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells; the epithelial cells include at least one selected from corneal epithelial cells, prostate epithelial cells, and renal tubular epithelial cells; the endothelial cells include at least one selected from pulmonary artery endothelial cells and aortic endothelial cells; the myocytes include at least one selected from aortic smooth muscle cells, pulmonary artery smooth muscle cells, and coronary artery smooth muscle cells; and the fibroblasts include at least one selected from cardiac fibroblasts, skin fibroblasts, and renal interstitial fibroblasts. Preferably, the above cells are mesenchymal stem cells.

[0111] According to an embodiment of the present invention, the stripping solution is a buffer solution free of calcium and magnesium ions. Therefore, the stripping solution is more effective in reducing the adhesion between the cell membrane and the culture dish. More preferably, the pH of the buffer solution free of calcium and magnesium ions is 6.9–7.4, for example, commercially available DPBS buffer can be used.

[0112] According to an embodiment of the present invention, a PVDF membrane can be used to collect monolayer cell films. Specifically, after the monolayer cell film detaches from the culture dish, a PVDF membrane can be used to adsorb the monolayer cell film onto the surface, and the film can be rinsed several times with physiological saline as needed. Preferably, the PVDF membrane carries a hydrophilically modified matrix, thereby further improving its adsorption effect on the monolayer cell film. More preferably, the PVDF membrane is a star-shaped hydrophilically modified PVDF membrane of dimethylaminoethyl acrylate polymer. Therefore, its adsorption effect on the monolayer cell film is even better.

[0113] Furthermore, according to embodiments of the present invention, reference Figure 2 and 3The PVDF membrane 300 includes one or more hollow regions. Therefore, the membrane surface has a certain amount of porosity, making the adsorption of monolayer cell membranes more convenient. In some embodiments of the present invention, the PVDF membrane is annular (e.g., Figure 2 (As shown).

[0114] Furthermore, according to embodiments of the present invention, the above-described method for preparing cell films may further include: stacking multiple monolayer cell films to obtain a composite cell film. Specifically, taking the stacking of two monolayer cell films as an example, two monolayer cell films can be collected using two PVDF membranes respectively, and then the two PVDF membranes can be bonded together with the cell film surfaces facing each other to obtain two stacked monolayer cell films.

[0115] According to an embodiment of the present invention, the prepared composite cell film may include 2 to 3 layers of cell film.

[0116] For ease of understanding, the following is a detailed description of the star-shaped PVDF membrane modified with dimethylaminoethyl acrylate polymer.

[0117] The star-shaped poly(dimethylaminoethyl acrylate) polymer (PDMAEA) has the structure shown in Formula I.

[0118]

[0119] In Equation I, R is n is a positive integer from 15 to 105. The degree of polymerization of this star-shaped dimethylaminoethyl acrylate polymer is between 100 and 400, and the molecular weight is between 10,000 and 60,000 Daltons. The inventors discovered that the long alkyl chains in the star-shaped dimethylaminoethyl acrylate polymer are hydrophobic and have good compatibility with PVDF; while the dimethylaminoethyl acrylate segments have high hydrophilicity, and the star-shaped structure also helps the star-shaped dimethylaminoethyl acrylate polymer to be more firmly fixed on the matrix membrane, thus improving the hydrophilicity and increasing the stability of the bond between the modifier and the PVDF membrane. Therefore, the PVDF membrane modified by the star-shaped dimethylaminoethyl acrylate polymer has higher hydrophilicity, permeability, and antifouling properties, and still has high recovery performance after multiple and long-term uses.

[0120] According to a specific embodiment of the present invention, the preparation method of star-shaped dimethylaminoethyl acrylate polymer and the preparation of PVDF membrane modified by star-shaped dimethylaminoethyl acrylate polymer hydrophilicity can be found in patent CN114797512A.

[0121] According to a specific embodiment of the present invention, a composite cell membrane is provided. According to an embodiment of the present invention, the composite cell membrane comprises: a plurality of stacked monolayer cell membranes, wherein the cells of the monolayer cell membranes are treated with a calcium- and magnesium-free buffer solution. Specifically, the monolayer cell membrane is composed of a bottom surface free energy not exceeding 90 mJ / m². 2 The cells were cultured in culture dishes and further treated with calcium- and magnesium-free buffer solutions. Therefore, the composite cell membrane preparation method of this invention is simple, efficient, and yields better therapeutic effects.

[0122] According to an embodiment of the present invention, the above-mentioned composite cell membrane comprises 2 to 3 layers of cell membrane.

[0123] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0124] Example 1: Preparation method of petri dish

[0125] The method for preparing the petri dish is as follows:

[0126] (1) Add 80 parts by weight of polystyrene particles, 20 parts by weight of butyl methacrylate and 10 parts by weight of poly(2-vinylpyridine-co-styrene) block copolymer to a reaction vessel in proportion, stir evenly, heat to 150-160℃, react for 10-12 hours, cool to room temperature (cooling rate not greater than 1.0℃ / min) to obtain mixture N1;

[0127] (2) Add 5 parts by weight of plasticizer cyclohexane-1,2-dicarboxylate diisooctyl ester and 2 parts by weight of anti-aging agent β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate to mixture N1, and knead at 500 rpm ± 20% for 10 min to obtain mixture N2.

[0128] (3) The hot-melt plasticized mixture N2 is put into an injection molding machine that has been preheated to 220°C (injection pressure is 80MPa (±10%)). Under the action of the screw, it is pressed into a hot-melt melt and injected into the culture dish molding mold. The pressure is held for 2-4 seconds, and the mold is opened after cooling to obtain the culture dish.

[0129] Example 2: Structure and characteristics of the petri dish

[0130] The culture dish was prepared using the method described in Example 1. Figure 4 As shown, the petri dish includes a petri dish body 1 and a protective cap 2. The petri dish body 1 is composed of a petri dish base plate 3 and a petri dish side wall. The protective cap 2 can be attached to the petri dish body 1, and a certain gap is left between the two to allow for gas exchange with the outside.

[0131] The petri dish body 1 and the antibacterial cap 2 are cylindrical. The diameter of the petri dish body 1 is 35 mm, and the actual culture area is approximately 8.8 cm². 2 The height is 13mm. The culture dish body 1 and the antibacterial cap 2 are made of polystyrene, ensuring high light transmittance for observing cell morphology during in vitro culture. The free energy of the bottom surface of the culture dish body 1 is no greater than 90mJ / m². 2 . refer to Figure 5 After thorough sterilization by irradiation, the culture dish was used for in vitro cell culture to prepare cell membranes. This example uses mesenchymal stem cells (MSCs). MSCs within the P5 level were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin / streptomycin, 1% glutamine, and 1% non-essential amino acids at 37°C and 5% carbon dioxide. When the cells reached confluence of 90% or more, they were digested with trypsin and seeded at a density of 10,000 cells / culture dish. The cells were then cultured under the same medium and conditions until confluence reached 95% or more. The medium was removed, and the cell membrane was washed with PBS. DPBS was then added to completely cover the cell membrane, and the dish was left at room temperature to allow the cell membrane to detach spontaneously. After replacing the medium with DPBS, the cell membrane completely detached from the culture dish within 15 minutes and floated in the DPBS solution as a complete monolayer. After washing the cell membrane several times with physiological saline, the cell membrane was ready for further research or application.

[0132] Example 3

[0133] Cell films were prepared using commercially available styrene culture dishes and culture dishes prepared by the method of the present invention in Example 1, respectively, using the same in vitro cell culture method as in Example 2. The integrity of the cell films after being peeled off from the bottom of the culture dishes was compared. Specifically, as follows... Figure 6 As shown in the figure, Figure A shows mesenchymal stem cells cultured in a commercially available styrene culture dish, with cells adhering and growing; Figure B shows the cell membrane ruptured after peeling using the method in Example 2; while the mesenchymal stem cells cultured in the culture dish provided by this invention show good cell adhesion and growth at the bottom of the culture dish. Figure 6 (C) can be naturally peeled off, and the cell membrane structure remains intact. Figure 6 (D).

[0134] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0135] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A petri dish, characterized in that, The materials of the petri dish include: (1) 75-80 parts by weight of polystyrene granules; (2) 10-40 parts by weight of polymer; (3) 1-5 parts by weight of plasticizer; (4) 1-5 parts by weight of anti-aging agent, The polymers include butyl methacrylate polymers and poly(2-vinylpyridine-co-styrene) block copolymers. The weight ratio of the butyl methacrylate polymer and the poly(2-vinylpyridine-co-styrene) block copolymer is 100:

50.

2. The petri dish according to claim 1, characterized in that, The plasticizer is selected from at least one of diisooctyl phthalate, neopentyl glycol dibenzoate, diisooctyl cyclohexane-1,2-dicarboxylate, diisononyl cyclohexane-1,2-dicarboxylate, and tributyl acetyl citrate. Optionally, the anti-aging agent is selected from at least one of pentaerythritol tetrakis[β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate], octadecyl β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate, dilauryl thiodipropionate (DLTP), and distearate thiodipropionate.

3. The petri dish according to claim 1, characterized in that, The surface free energy of the culture dish does not exceed 90 mJ / m 2 .

4. The petri dish according to claim 1, characterized in that, The surface free energy of the culture dish does not exceed 60 mJ / m 2 .

5. A method for preparing a petri dish, characterized in that, The petri dish is made from the following raw materials: (1) 75-80 parts by weight of polystyrene granules; (2) 10-40 parts by weight of polymer; (3) 1-5 parts by weight of plasticizer; (4) 1-5 parts by weight of anti-aging agent, The polymers include butyl methacrylate polymers and poly(2-vinylpyridine-co-styrene) block copolymers. The weight ratio of the butyl methacrylate polymer and the poly(2-vinylpyridine-co-styrene) block copolymer is 100:

50.

6. The preparation method according to claim 5, characterized in that, The plasticizer is selected from at least one of diisooctyl phthalate, neopentyl glycol dibenzoate, diisooctyl cyclohexane-1,2-dicarboxylate, diisononyl cyclohexane-1,2-dicarboxylate, and tributyl acetyl citrate. Optionally, the anti-aging agent is selected from at least one of pentaerythritol tetrakis[β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate], octadecyl β-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate, dilauryl thiodipropionate, and distearate thiodipropionate.

7. The preparation method according to claim 5, characterized in that, The pressing process includes: (A) The polystyrene particles and polymer are mixed and heated at 150-160°C for 10-12 h to obtain the first mixture M1; (B) Add the plasticizer and anti-aging agent to the first mixture M1, knead, and obtain the second mixture M2; (C) The second mixture M2 is injection molded to obtain the culture dish.

8. The preparation method according to claim 7, characterized in that, The pressing process further includes: Before proceeding to step (B), the first mixture M1 is cooled down to 20-30°C. Optionally, the cooling rate is no greater than 1.0℃ / min; Optionally, in step (B), the kneading speed is 400-600 rpm and the time is 5-15 min.

9. The preparation method according to claim 7, characterized in that, In step (C), the injection molding process includes: (a) The second mixture M2 is fed into an injection molding machine and extruded into a hot melt state, wherein the barrel temperature of the injection molding machine is 200-250°C; (b) The hot-molten melt is injected into the mold and held under pressure for 2-4 seconds to obtain the culture dish.

10. Use of the culture dish according to any one of claims 1-4 or the culture dish prepared by the preparation method according to any one of claims 5-9 in the preparation of cell films.

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