CN114806869A - Culture dish and application thereof in preparing cell film - Google Patents

Culture dish and application thereof in preparing cell film Download PDF

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Publication number
CN114806869A
CN114806869A CN202110129346.1A CN202110129346A CN114806869A CN 114806869 A CN114806869 A CN 114806869A CN 202110129346 A CN202110129346 A CN 202110129346A CN 114806869 A CN114806869 A CN 114806869A
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methacrylate
cells
acrylate
culture dish
cell
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孙佳
郭啸华
唐漫书
廖鑫
胡隽源
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Shenzhen Carbon Source Biotechnology Co ltd
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Shenzhen Carbon Source Biotechnology Co ltd
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Abstract

The invention discloses a culture dish and application thereof in preparing a cell film. Wherein, the culture dish includes: a body defining a culture space therein; and a coating disposed at the bottom of the culture space, wherein the coating has a surface free energy of no more than 90mJ/m 2 . Therefore, the binding force between the coating and the cells can be reduced by adjusting the ion concentration in the cell liquid culture medium, so that the cells cultured by adherence can fall off from the coating at the bottom of the culture dish in the form of a cell film, and the cell film is effectively obtained.

Description

Culture dish and application thereof in preparing cell film
Technical Field
The invention relates to the field of biomedicine, in particular to the field of bioengineering, in particular to a culture dish and application thereof in preparing a cell film, and particularly relates to the culture dish, the application of the culture dish in preparing the cell film, a method for adjusting binding force of cells and the culture dish, a method for preparing the cell film, a composite cell film, the application of the composite cell film in preparing a medicament and a pharmaceutical composition.
Background
With the development of biology and tissue engineering technology, biodegradable medical polymer implants and polymer-cell chimeric implants have gained wide attention in the fields of injury repair, tissue regeneration, organ transplantation, and the like. However, the immune reaction brought by the degradable biopolymer material and the inflammatory reaction brought by the degradable biopolymer material after decomposition limit the clinical application of the degradable biopolymer material; other methods have obvious disadvantages, such as mixing the cell suspension and the polymer solution and then infusing, and the cell regeneration efficiency and utilization rate after the back infusion are often low because the extracellular matrix (ECM) is damaged by hydrolytic enzyme. To solve the above problems, cell films (cell sheets) have attracted much attention as a cell material without foreign materials in cell transplantation applications.
The cell membrane is a complete monolayer cell membrane obtained by culturing cells obtained by separation on a specific material under in vitro culture conditions and separating the cells from a culture substrate by a specific means without destroying cell connection. Compared with cell injection and polymer-cell chimeric implants, the cell membrane has the following advantages in clinical application: (1) after the traditional cells are injected into veins/arteries, the distribution of the cells is biased to tissues such as liver, lung and the like, and the mesenchymal stem cells are difficult to target to transplanted organ parts; and the cell membrane can be directly transplanted to a required position in a transplantation operation. (2) The traditional cell system injection is often difficult to achieve good curative effect on diseases of certain special parts, such as bone joints, tendons and the like, due to the lack of abundant capillary networks in tissues and the like; the cell membrane can be directly applied to the parts to play a key therapeutic role. (3) The cell membrane does not need to be supported by degradable high polymer materials, so that immune reaction and inflammatory reaction caused by the high polymer materials are not easy to initiate. (4) The cell membrane completely reserves extracellular matrix, and is favorable for regeneration and in-vivo utilization of cells.
The current technology for preparing cell membranes is mainly based on temperature-sensitive high polymer non-adhesive cell culture vessels developed by Teruo Okano et al. However, this preparation method has significant drawbacks: for example, the change of the temperature causes the nonuniform change of the surface chemical properties of the culture dish, the time consumption for preparing the cell film is long, the preparation method is difficult, and the like; other preparation methods such as an ultrasonic stimulation method, an electrical stimulation method, an ultraviolet irradiation method and the like have defects. Therefore, the current means for preparing cell membranes still need to be improved.
Disclosure of Invention
The present invention is directed to solving, at least in part, one of the technical problems in the related art. Therefore, an object of the present invention is to provide a means for efficiently separating adherent cells from a culture dish by adjusting the ion concentration to obtain a cell thin film.
In a first aspect of the invention, a culture dish is provided. According to an embodiment of the invention, the culture dish comprises: a body defining a culture space therein; and a coating disposed at the bottom of the culture space, wherein the coating has a surface free energy of no more than 90mJ/m 2 。
According to the embodiment of the present invention, when cells such as stem cells and the like are cultured using the culture dish, particularly when the culture is performed using a liquid medium, the cells can adhere to the coating layer at the bottom of the culture space during the expansion process and can be proliferated adherently on the coating layer, and further, in view of the fact that the surface free energy of the coating layer does not exceed 90mJ/m 2 By adjusting the ion concentration in the cell liquid culture medium, the binding force between the coating and the cells can be reduced, so that the cells cultured by adherence can fall off from the coating at the bottom of the culture dish in the form of a cell film, and the cell film is effectively obtained. The inventors have surprisingly found that detachment of the cell membrane from the bottom of the culture dish can be accomplished rapidly by this method, typically by not more than 1 hour, for example not more than 30 minutes, such as not more than 20 minutes, for example 15 minutes after changing the ion concentration. The reason for this is probably because the cell adhesion plaque complex crosses the membrane by changing the ion concentration in the medium, for example, the concentration of calcium ion and/or magnesium ionThe configuration of the receptor can be changed, and the binding force between the coating and the cells can be reduced, so that the cells can be promoted to be separated from the bottom surface of the culture dish under the condition that the proteins among the cells are not hydrolyzed by trypsin, and a complete cell film is obtained.
According to an embodiment of the invention, the culture dish may further have at least one of the following additional technical features:
according to an embodiment of the invention, the thickness of the coating does not exceed 300 nm. According to the embodiments of the present invention, the inventors found that if the thickness of the coating exceeds 300nm, the growth of cells is adversely affected.
According to an embodiment of the invention, the thickness of the coating is not less than 2nm, but not more than 100nm, preferably not more than 50nm, more preferably not more than 30nm, most preferably not more than 10 nm. Thus, the effect of detaching the cell membrane from the culture dish is better.
According to an embodiment of the invention, the surface free energy of the coating does not exceed 60mJ/m 2 . Thus, the effect of detaching the cell membrane from the culture dish is better.
According to an embodiment of the invention, the coating contains a high polymer or block copolymer formed from at least one of the following monomers: vinylimidazole (vinylimidazole), vinylpyrrolidone (vinylirrolidone), aminostyrene (aminostyrene), methacrylamide (methacrylamide), dimethylacrylamide (N, N-dimethylacrylamide), N-isopropylacrylamide (N-isopropylacrylamide), 4-vinylbenzyl chloride (4-vinylbenzyl chloride), cyanomethylstyrene (vinylinyl cyanide), 1-methylpyridine (1-methylpyridium chloride), vinylcaprolactam (N-vinylpropylacrylamide), acrylic acid (acrylic acid), N-dimethylaminoethyl acrylate (dimethylaminoethyl acrylate), chloroethyl acrylate (propylvinyl acetate), cyanoethylmethacrylate (cyclohexylacrylate), 3- (3-dimethylaminopropyl) acrylate (3- (2-propylmethacrylate), styrene (2-isopropylstyrene), vinylbenzyl chloride (2-isopropylstyrene), cyclohexylmethacrylate (3-isopropylmethacrylate), cyclohexylmethacrylate (2-isopropylstyrene (2-propylene), and (2-isopropylstyrene (2-propylene-ethylene-2-propylene-acrylate), and (2-propylene-2-propylene-ethylene) acrylate (2-propylene-ethylene-propylene-2-propylene-ethylene-2-propylene-acrylate) Acrylonitrile (acrylonitrile), caprolactone (caprolactone), N-dimethylvinylbenzylmethylamine (dimethylvinylstyrene), 4-vinylpyridine (4-vinylpyridine), divinylbenzene (dimethylbenzene), vinyl Benzoate (vinyl Benzoate), benzyl methacrylate (benzyl methacrylate), cyclohexyl methacrylate (cyclohexyl methacrylate), butyl methacrylate (butyl methacrylate), isopropyl methacrylate (isopropyl methacrylate), acrylamide (acrylate), allyl methacrylate (allyl methacrylate), isocyanuric ethyl methacrylate (2-isocyanatomethacrylate), ethylene glycol dimethacrylate (ethylene glycol dimethacrylate), ethylene glycol dimethacrylate (hydroxyethyl methacrylate), hydroxyethyl methacrylate (2-isocyanatomethacrylate), polymethyl methacrylate (1-hydroxyethyl methacrylate), polymethyl methacrylate (4-dimethylolmethacrylate), methyl methacrylate (4-hydroxyethyl methacrylate), methyl methacrylate (2-isocyanatomethacrylate), methyl methacrylate (4-hydroxyethyl methacrylate), methyl methacrylate (1-hydroxyethyl methacrylate), methyl methacrylate (2-isocyanatomethacrylate), methyl methacrylate (hydroxyethyl methacrylate), methyl methacrylate (4-hydroxyethyl methacrylate), methyl methacrylate (2-vinylbenzyl methacrylate), methyl methacrylate (4-hydroxyethyl methacrylate), methyl methacrylate (4-vinylbenzyl methacrylate), methyl methacrylate (4-hydroxyethyl methacrylate), methyl methacrylate (2-vinylbenzyl methacrylate), methyl methacrylate (cyclohexyl methacrylate), methyl methacrylate (methyl methacrylate), methyl methacrylate, cyclohexyl methacrylate (4-hydroxyethyl methacrylate), methyl methacrylate (methyl methacrylate), methyl methacrylate, methyl acrylate, methyl methacrylate, methyl acrylate, methyl methacrylate, Tetrahydrofurfuryl methacrylate (tetrahydrofurfuryl methacrylate), hexyl methacrylate (hexyl methacrylate), hydroxyethyl methacrylate (hydroxyethyl methacrylate), glycidyl methacrylate (glycidyl methacrylate), propynyl methacrylate), 1,4-butanediol vinyl ether (1, 4-butandiol dimethacrylate), isobornyl acrylate (isobornyl acrylate), ethylene glycol diacrylate (ethylene glycol diacrylate), propynyl acrylate (propynyl acrylate), 2,4,6,8-tetramethyl-2,4,6, 8-tetraoxane (2,4,6, 8-tetravinylcyclotetrasiloxane) (2,4,6, 8-tetramethylenedioxane), hexavinyldisiloxane (hexasiloxane), hexavinyldisiloxane (1,4, 6, 4-trimethylcyclosiloxane) (1,4, 5, 1,4, 6, 4-trimethylcyclomethicone), 3, 5-trimethylchlorotrisiloxane), trivinyltrimethylcyclotrisilazane (2,4, 6-trimethylcyclotrisilazane), dimethylphenylvinylsilane (dimethylphenylvinylsilane), (perfluorooctyl) ethylmethacrylate (perfluorooctyl) perfluorodecyl methacrylate), perfluorodecyl acrylate (perfluorodecyl acrylate), heptafluorobutyl methacrylate (heptafluorobutyl methacrylate), 1,1,1,3,3,3-hexafluoroisopropylmethacrylate (1,1,1,3,3,3-hexafluoroisopropylmethacrylate), 2,3,3,4,4-hexafluoro-1, 5-pentaacrylate (2,2,3,3,4,4-hexafluoro-1, 5-pentaacrylate), 2,3, 4,4-hexafluoro-1, 5-pentaacrylate (2, 5-perfluorohexyl methacrylate), 2- (2, 2-trifluoroethylhexyl) ethyl methacrylate (2,2,3, 4, 4-trifluoromethylcyclohexyl) and 2,2,3, 4-trifluoromethylcyclohexyl (2, 4-trifluoroethylhexyl methacrylate), 2-trifluoroethoxy methacrylate, pentafluorophenyl methacrylate, 1H,7H-dodecafluoro heptyl acrylate (1H,1H,7H-dodecafluoro heptyl acrylate), 1H,2H, 2H-perfluorodecyl acrylate (1H,1H,2H, 2H-heptafluoro alkenyl acrylate), diethylene glycol divinyl ether (diethylene glycol divinyl ether), 1,9-decadiene (1,9-decadiene), 2-methacrylic anhydride (methacrylic anhydride), 1,2,4-trivinylcyclohexane (1,2,4-trivinylcyclohexane), acetoacetic acid (allyl ester), maleic anhydride (maleic anhydride), 1,2,4-trivinylcyclohexane (1,2, 4-trifluoromethylcarbazole), 2-cyanoaniline (9-cyanoethyl acrylate), 2-trifluoromethylaniline (4-trifluoromethylcarbazole), 2-cyanoaniline (9-cyanoaniline), 2-1, 4-trifluoromethylaniline (9-cyanoaniline (9-cyanoaniline), 2-1, 4-dodecylaniline (9-cyanoaniline (9-cyanoaniline), 2, 4-1, 4-bis (9-cyanoaniline, 2-cyanoaniline (9-2-cyanoaniline, 4-one, 2-one, one, N, N-diethylaminoethyl acrylate (diethylaminoethyl acrylate), 2- (dimethylamino) ethyl acrylate (dimethylamino) ethyl 3- (dimethylamino) propyl acrylate (3- (dimethylamino) propyl acrylate), 2- (dimethylamino) ethyl methacrylate (2- (dimethylamino) ethyl methacrylate), 2- (tert-butylamino) ethyl methacrylate (t-butylamino) ethyl methacrylate), N-dimethylvinylbenzylamine (dimethylvinylbenzylstyrene), methacrylic acid (methacrylic acid), acrylamide (acrylamide), vinyl-N-methylpyridinium chloride (vinyl-N-methylpyridinium chloride), N-dimethylvinylbenzylamine (N- (4-vinylbenzyl) -N, N-dimethylvinylbenzylamine (N-dimethylvinylbenzyl).
According to an embodiment of the invention, the coating is formed from a poly (2-vinylpyridine-co-styrene) block copolymer. Thus, the effect of detaching the cell membrane from the culture dish is better.
According to an embodiment of the present invention, the coating layer is formed by at least one of a solution evaporation method, a spray coating method, a vacuum evaporation coating method, a chemical vapor deposition method, a pulsed laser deposition method, and a sol-gel method.
According to an embodiment of the invention, the coating is formed by solid phase deposition of 2-vinylpyridine and styrene.
According to an embodiment of the invention, the culture dish further comprises: a bottom plate detachably provided at the bottom of the culture space, wherein the coating layer is provided on a surface of the bottom plate.
In a second aspect of the invention, the invention proposes the use of the culture dish of the above example for the preparation of a thin film of cells. As mentioned above, the culture space of the medium has a free energy at the bottom of the culture space of not more than 90mJ/m 2 Coating of (2). Therefore, the binding force between the coating and the cells can be reduced by adjusting the ion concentration in the cell liquid culture medium, so that the cells cultured by adherence can fall off from the coating at the bottom of the culture dish in the form of a cell film, and the cell film is effectively obtained.
In a third aspect of the invention, a method of adjusting binding of a cell to a culture dish is provided. According to an embodiment of the invention, the culture dish is the culture dish of the above embodiment, the method comprises: the concentration of calcium and/or magnesium ions in the cell culture fluid is altered. The inventors have surprisingly found that by varying the ion concentration in the medium, for example the concentration of calcium and/or magnesium ions, the conformation of the cell adhesion plaque complex transmembrane receptor is altered, whereby the binding force between the coating and the cells is reduced. Further, by this method, a cell membrane can be efficiently produced.
In a fourth aspect of the invention, a method of preparing a thin film of cells is presented. According to an embodiment of the invention, the method comprises: culturing cells in the culture dish of the above example using a liquid medium to form a monolayer cell thin film; subjecting the monolayer cell film to a stripping solution having a lower concentration of cations than the liquid medium, the cations comprising at least one of calcium and/or magnesium ions; collecting the monolayer cell film from the stripping solution.
According to the embodiment of the present invention, when cells such as stem cells and the like are cultured using the culture dish of the above embodiment, the cells can adhere to the coating layer on the bottom of the culture space during the expansion process and can be proliferated adherent to the coating layer, and further, in view of that the surface free energy of the coating layer in the culture dish does not exceed 90mJ/m 2 The ion concentration in the culture medium is adjusted by utilizing the fact that the calcium ion and/or magnesium ion concentration is lower than the stripping of the liquid culture medium, so that the binding force between the coating and the cells can be reduced, the cells cultured by adherence can fall off from the coating at the bottom of the culture dish in the form of a cell film, and the cell film is effectively obtained. The inventors have surprisingly found that detachment of the cell membrane from the bottom of the culture dish can be accomplished rapidly by this method, typically by not more than 1 hour, for example not more than 30 minutes, such as not more than 20 minutes, for example 15 minutes after changing the ion concentration. The reason for this is probably because the configuration of the cell adhesion plaque complex transmembrane receptor is changed by changing the concentration of calcium ions and/or magnesium ions in the culture medium, and the binding force between the coating and the cells is reduced, so that the cells can be promoted to be separated from the bottom surface of the culture dish without trypsinizing the intercellular proteins, and a complete cell membrane can be obtained.
According to an embodiment of the present invention, the method for preparing a cell membrane may further have at least one of the following additional technical features:
according to an embodiment of the invention, the cell comprises at least one selected from the group consisting of stem cells, neuronal cells, astrocytes, oligodendrocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts.
According to an embodiment of the present invention, the stem cell includes at least one of an induced pluripotent stem cell, an adult stem cell, a mesenchymal stem cell, a neural stem cell, a cardiac stem cell and a lung stem cell.
According to an embodiment of the present invention, the mesenchymal stem cell includes at least one of a bone marrow-derived mesenchymal stem cell, an adipose-derived mesenchymal stem cell, and an umbilical cord-derived mesenchymal stem cell.
According to an embodiment of the invention, the epithelial cells comprise at least one of corneal epithelial cells, prostate epithelial cells, renal tubular epithelial cells, coronary artery.
According to an embodiment of the invention, the endothelial cells comprise at least one of vascular endothelial cells, pulmonary arterial endothelial cells, aortic endothelial cells.
According to an embodiment of the invention, the muscle cells comprise at least one of aortic smooth muscle cells, pulmonary smooth muscle cells, coronary smooth muscle cells.
According to an embodiment of the invention, the fibroblasts comprise at least one of cardiac fibroblasts, skin fibroblasts, renal interstitial fibroblasts, preferably mesenchymal stem cells.
According to an embodiment of the present invention, the stripping solution is a buffer solution that does not contain calcium ions and magnesium ions.
According to the embodiment of the invention, the pH value of the buffer solution is 6.9-7.4, and preferably, the buffer solution is DPBS.
According to an embodiment of the present invention, the single-layer cell thin film is collected using a PVDF membrane.
According to an embodiment of the present invention, the PVDF membrane carries a hydrophilic modification group, and preferably, the PVDF membrane is a star-poly (dimethylaminoethyl acrylate) polymer hydrophilic modification PVDF membrane.
According to an embodiment of the invention, the PVDF membrane comprises a hollow region.
According to an embodiment of the invention, the PVDF membrane is circular.
According to an embodiment of the invention, the method further comprises: and superposing a plurality of the monolayer cell films so as to obtain a composite cell film.
According to the embodiment of the invention, the composite cell film comprises 2-3 layers of cell films.
In a fifth aspect of the invention, the invention provides a composite cell membrane. According to an embodiment of the present invention, the composite cell membrane includes: a plurality of monolayer cell membranes stacked, wherein cells of the monolayer cell membranes are treated with a calcium-magnesium free buffer. Specifically, the monolayer cell film is formed by a material with free energy not more than 90mJ/m 2 Culturing in coated culture medium, and further treating with calcium-free magnesium buffer solution. Therefore, the preparation method of the composite cell film is simple, convenient and efficientAnd the treatment effect is better.
According to an embodiment of the present invention, the composite cell membrane may further have at least one of the following additional features:
according to an embodiment of the invention, the composite cell membrane comprises 2-3 cell membranes.
According to an embodiment of the present invention, the monolayer cell membrane is formed by the method for preparing a cell membrane of the above embodiment.
In a sixth aspect of the invention, the invention provides the use of the composite cell membrane of the above embodiment in the preparation of a medicament. According to an embodiment of the invention, the medicament is for the treatment of at least one selected from the group consisting of: ocular injury repair and regeneration, skin and soft tissue necrosis, repair of wounded tissue, skin burn, cold injury, kidney transplantation, liver transplantation, heart transplantation, lung transplantation, soft tissue transplantation, wound repair, cosmetic surgery, abdominal hernia repair, liver injury, spleen rupture, stomach injury, duodenal injury, small intestine and mesenteric injury, gastric ulcer, duodenal ulcer, diabetic foot, central nervous system injury repair and regeneration, craniocerebral injury, spinal cord injury, myocarditis, myocardial infarction, atrial septal defect, ventricular septal defect, fracture healing, joint injury, or development of artificial bone. As described above, the method for preparing the composite cell membrane of the above embodiments is simple and efficient, and the therapeutic effect is better. Therefore, the composite cell film is used for preparing medicines, and a better treatment effect can be obtained.
In a seventh aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: the composite cell membrane of the above example; and pharmaceutically acceptable adjuvants. As described above, the method for preparing the composite cell membrane of the above embodiments is simple and efficient, and the therapeutic effect is better. Therefore, the composite cell membrane is used for the pharmaceutical composition, and a better treatment effect can be obtained.
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.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a schematic diagram of a culture dish according to one embodiment of the invention;
FIG. 2 is a schematic structural view of a culture dish according to yet another embodiment of the invention;
FIG. 3 is a schematic structural view of a PVDF membrane according to one embodiment of the invention;
FIG. 4 is a schematic structural view of a PVDF membrane according to yet another embodiment of the invention;
FIG. 5 is a NMR spectrum of a star-polydimethylaminoethyl polyacrylate polymer according to one embodiment of the present invention;
FIG. 6 is an SEM image of a star-polydimethylaminoethyl polyacrylate polymer according to one embodiment of the present invention;
FIG. 7 is a water contact angle test plot of a star-polydimethylaminoethyl acrylate polymer hydrophilically modified PVDF membrane in accordance with one embodiment of the present invention;
FIG. 8 is a schematic structural diagram of a composite cell membrane according to one embodiment of the present invention;
FIG. 9 is a schematic view of the structure of a culture dish in example 1;
FIG. 10 is a schematic view of the preparation and application of a culture dish in example 1;
FIG. 11 is a schematic view showing the principle of separating the cell membrane from the culture dish under the condition of changing the concentrations of calcium and magnesium ions in example 1;
FIG. 12 is a graph showing the effect of peeling the cell membrane from the culture dish in example 1.
Reference numerals:
100: a body;
110: a culture space;
200: coating;
120: a base plate;
300: a PVDF membrane;
40: a monolayer cell membrane;
1: a cell-bearing dish;
2: a bacteria-proof cap;
3: poly (2-vinylpyridine-co-styrene) block copolymer coatings.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on those shown in the drawings, and are used only for convenience in describing the present invention and for simplicity in description, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed in a particular orientation, and be operated, and thus, are not to be construed as limiting the present invention.
Furthermore, the terms "first", "second", etc. are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first," "second," etc. may explicitly or implicitly include at least one of the feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.
In a first aspect of the invention, a culture dish is provided. Referring to fig. 1, according to an embodiment of the present invention, the culture dish includes: a body 100 and a coating 200. The body 100 defines a culture space 110 therein, and the coating 200 is disposed at the bottom of the culture space 110. Wherein the surface free energy of the coating 200 is not more than 90mJ/m 2 . More preferably, the surface free energy of the coating 200 does not exceed 60mJ/m 2 . Thus, the effect of detaching the cell membrane from the culture dish is better.
The specific shape of the culture dish body is not particularly limited, and can be selected by those skilled in the art according to actual needs. According to some embodiments of the present invention, the overall shape of the culture dish is circular, the cell bearing surface is smooth and flat, the height is 13-17 cm, the diameter is 35-100 mm, and the effective cell culture area is 8-57 cm 2 。
In addition, the specific material of the culture dish body is not particularly limited, and is preferably a polystyrene substrate. Therefore, the culture dish body has good light transmittance, and the cell morphology is convenient to observe. In some embodiments of the invention, the transmittance of the culture dish body to visible light with a wavelength of 400-800 nm is more than 80%.
According to an embodiment of the invention, the thickness of the coating 200 does not exceed 300 nm. The inventors have unexpectedly discovered in their studies that the thickness of the coating 200 is too great and does not facilitate the growth of cells thereon. Further, the thickness of the coating 200 is not less than 2nm, but not more than 100nm, preferably not more than 50nm, more preferably not more than 30nm, and most preferably not more than 10 nm. Thus, the effect of detaching the cell membrane from the culture dish is better.
According to an embodiment of the invention, the coating 200 contains a high polymer or block copolymer formed from at least one of the following monomers: vinylimidazole (vinylimidazole), vinylpyrrolidone (vinylirrolidone), aminostyrene (aminostyrene), methacrylamide (methacrylamide), dimethylacrylamide (N, N-dimethylacrylamide), N-isopropylacrylamide (N-isopropylacrylamide), 4-vinylbenzyl chloride (4-vinylbenzyl chloride), cyanomethylstyrene (vinylinyl cyanide), 1-methylpyridine (1-methylpyridium chloride), vinylcaprolactam (N-vinylpropylacrylamide), acrylic acid (acrylic acid), N-dimethylaminoethyl acrylate (dimethylaminoethyl acrylate), chloroethyl acrylate (propylvinyl acetate), cyanoethylmethacrylate (cyclohexylacrylate), 3- (3-dimethylaminopropyl) acrylate (3- (2-propylmethacrylate), styrene (2-isopropylstyrene), vinylbenzyl chloride (2-isopropylstyrene), cyclohexylmethacrylate (3-isopropylmethacrylate), cyclohexylmethacrylate (2-isopropylstyrene (2-propylene), and (2-isopropylstyrene (2-propylene-ethylene-2-propylene-acrylate), and (2-propylene-2-propylene-ethylene) acrylate (2-propylene-ethylene-propylene-2-propylene-ethylene-2-propylene-acrylate) Acrylonitrile (acrylonitrite), caprolactone (caprolactone), N-dimethylvinylbenzene methylamine (dimethylvinyltoluene), 4-vinylpyridine (4-vinylpyridine), divinylbenzene (dimethylbenzene), vinyl Benzoate (vinyl Benzoate), benzyl methacrylate (benzyl methacrylate), cyclohexyl methacrylate (cyclohexylmethacrylate), butyl methacrylate (butyl methacrylate), isopropyl methacrylate (isopropylmethacrylate), acrylamide (acrylamide), allyl methacrylate (allyl methacrylate), isocyano methacrylate (2-isocyanatoethyl methacrylate), ethylene glycol dimethacrylate (ethylene glycol dimethacrylate), ethylene glycol methacrylate (ethylene glycol methacrylate), ethylene glycol methacrylate (4-hydroxyethyl methacrylate), methyl methacrylate (1, 4-hydroxyethyl methacrylate), methyl methacrylate (2-hydroxyethyl methacrylate), and the like, Tetrahydrofurfuryl methacrylate (tetrahydrofurfuryl methacrylate), hexyl methacrylate (hexylmethacrylate), hydroxyethyl methacrylate (hydroxyethyl methacrylate), glycidyl methacrylate (glycidyl methacrylate), propynyl methacrylate (propylmethacrylate), 1,4-butanediol vinyl ether (1, 4-butandiol ether), isobornyl acrylate (isobornyl acrylate), ethylene glycol diacrylate (ethylene glycol diacrylate), propynyl acrylate (propylacrylate), 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane (2,4,6, 8-tetracyclotetrasiloxane), hexavinyldisiloxane (2,4,6, 8-trimethylcyclotetrasiloxane), hexavinyldisiloxane (1,4, 5, 3, 5-trivinylcyclotetrasiloxane), 3, 5-trimethylchlorotrisiloxane), trivinyltrimethylcyclotrisilazane (2,4, 6-trimethylcyclotrisilazane), dimethylphenylvinylsilane (dimethylphenylvinylsilane), (perfluorooctyl) ethylmethacrylate (perfluorooctyl) perfluorodecyl methacrylate), perfluorodecyl acrylate (perfluorodecyl acrylate), heptafluorobutyl methacrylate (heptafluorobutyl methacrylate), 1,1,1,3,3,3-hexafluoroisopropylmethacrylate (1,1,1,3,3,3-hexafluoroisopropylmethacrylate), 2,3,3,4,4-hexafluoro-1, 5-pentaacrylate (2,2,3,3,4,4-hexafluoro-1, 5-pentaacrylate), 2,3, 4,4-hexafluoro-1, 5-pentaacrylate (2, 5-perfluorohexyl methacrylate), 2- (2-trifluoroethylhexyl) ethyl methacrylate (2,2,3, 4, 4-trifluoromethylcyclohexyl) and 2- (perfluorohexyl) acrylate (2,2,3,3,4,4-hexafluoro-1, 5-pentaacrylate), 2, 2-trifluoroethoxy methacrylate), pentafluorophenyl methacrylate, 1H,7H-dodecafluoroheptyl acrylate (1H,1H,7H-dodecafluoroheptyl acrylate), 1H,2H, 2H-perfluorodecyl acrylate (1H,1H,2H, 2H-heptafluoro acrylate), diethylene glycol divinyl ether (diethylene glycol divinyl ether), 1,9-decadiene (1,9-decadiene), 2-methacrylic anhydride (methacrylic anhydride), 1,2,4-trivinylcyclohexane (1,2,4-trivinylcyclohexane), acetoacetic acid allyl ester (allyl acetate), maleic anhydride (maleic anhydride), 2-vinylcarbazole (4-vinylcarbazide), 2-cyanoaniline (4-vinylcarbazole), 2-cyanoaniline (9-cyanoethyl acrylate), 2-cyanoaniline (4-cyanoaniline (2-9-cyanoaniline), 2, 4-vinylcarbazole (9-cyanoaniline), 2, 9-cyanoaniline (9-cyanoaniline), 1, 9-cyanoaniline (allyl acetate), 2-cyanoaniline (9-cyanoaniline), 2, 4-cyanoaniline (9-cyanoaniline (9-cyanoaniline), 2, 4-cyanoaniline (9-cyanoaniline), 2, 4-cyanoaniline (9-cyanoaniline, 2-1, 2-cyanoaniline, 2-one, 2-cyanoaniline, 2-one, 2, and (4-one, 2-one, 4-one, 2-one, one, N, N-diethylaminoethyl acrylate (dimethylamino) ethyl acrylate, 3- (dimethylamino) propyl acrylate (3- (dimethylamino) propyl acrylate), 2- (dimethylamino) ethyl methacrylate (2- (dimethylamino) ethyl methacrylate), 2- (t-butylamino) ethyl methacrylate), N-dimethylvinylbenzylamine (dimethylvinylbenzylstyrene), methacrylic acid (methacrylic acid), acrylamide (acrylamide), vinyl-N-methylchloride (vinyl-N-methylpyridinium chloride), N-dimethylvinylbenzylamine (N- (4-vinylbenzyl) -N, N-dimethylvinylbenzylamine).
Preferably, the coating 200 is formed of poly (2-vinylpyridine-co-styrene) block copolymer and polyvinylbenzene, wherein the ratio of poly (2-vinylpyridine-co-styrene) block copolymer to polyvinylbenzene may be (50-100): 1,. More preferably, the coating 200 is formed entirely of poly (2-vinylpyridine-co-styrene) block copolymer.
In addition, the formation manner of the coating layer 200 is not particularly limited, and may be selected by those skilled in the art according to actual needs. According to some embodiments of the present invention, the coating layer 200 may be formed by at least one of a solution evaporation method, a spray coating method, a vacuum evaporation coating method, a chemical vapor deposition method, a pulsed laser deposition method, and a sol-gel method. When the material of the coating 200 is selected to be a poly (2-vinylpyridine-co-styrene) block copolymer, it is preferably formed by solid phase deposition of 2-vinylpyridine and styrene.
According to an embodiment of the present invention, referring to fig. 2, the culture dish of the present invention may further comprise: a base plate 120. The base plate 120 is detachably provided at the bottom of the culture space 110, wherein the coating layer 200 is provided on the surface of the base plate 120. This can further improve the convenience of taking out the cell thin film formed on the surface of the coating layer 200 from the culture dish.
In a second aspect of the invention, the invention proposes the use of the culture dish of the above example for the preparation of a thin film of cells. As mentioned above, the culture space of the medium has a free energy at the bottom of the culture space of not more than 90mJ/m 2 Coating of (2). Therefore, the binding force between the coating and the cells can be reduced by adjusting the ion concentration in the cell liquid culture medium, so that the cells cultured by adherence can fall off from the coating at the bottom of the culture dish in the form of a cell film, and the cell film is effectively obtained.
In a third aspect of the invention, a method of adjusting binding of a cell to a culture dish is provided. According to an embodiment of the invention, the culture dish is the culture dish of the above embodiment, the method comprises: the concentration of calcium and/or magnesium ions in the cell culture fluid is altered. The inventors have surprisingly found that by varying the ion concentration in the medium, for example the concentration of calcium and/or magnesium ions, the conformation of the cell adhesion plaque complex transmembrane receptor is altered, whereby the binding force between the coating and the cells is reduced. Further, by this method, a cell membrane can be efficiently produced.
In a fourth aspect of the invention, a method of preparing a thin film of cells is presented. According to an embodiment of the invention, the method comprises: culturing cells in the culture dish of the above example using a liquid medium to form a monolayer cell thin film; placing the monolayer cell film in a stripping solution, wherein the concentration of cations in the stripping solution is lower than that of the liquid culture medium, and the cations comprise at least one of calcium ions and/or magnesium ions; from the stripping solution, a monolayer cell film was collected.
The method for preparing a cell membrane according to an embodiment of the present invention is further described in detail below.
The method for preparing a cell membrane according to the present invention is not particularly limited with respect to the specific type of cells, and may be, for example, at least one selected from stem cells, neuronal cells, astrocytes, oligodendrocytes, epithelial cells, endothelial cells, muscle cells, and fibroblasts. Specifically, the stem cell includes at least one of an induced pluripotent stem cell, an adult stem cell, a mesenchymal stem cell, a neural stem cell, a cardiac stem cell and a lung stem cell; the mesenchymal stem cells comprise at least one of 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 of corneal epithelial cells, prostate epithelial cells, renal tubular epithelial cells, and coronary artery cells; the endothelial cells comprise at least one of endothelial cells of arteries, pulmonary artery and aorta; the muscle cells include at least one of aortic smooth muscle cells, pulmonary smooth muscle cells, and coronary smooth muscle cells; the fibroblast includes at least one of myocardial fibroblast, skin fibroblast and renal interstitial fibroblast. Preferably, the above cells are mesenchymal stem cells.
According to an embodiment of the present invention, the stripping solution is a buffer solution containing no calcium ion and no magnesium ion. Therefore, the stripping solution has better effect of reducing the binding force between the cell membrane and the culture dish. More preferably, the buffer solution without calcium ions and magnesium ions has a pH of 6.9 to 7.4, and for example, a commercially available DPBS buffer solution can be used.
According to embodiments of the present invention, a PVDF membrane may be used to collect a monolayer of a cell membrane. Specifically, after the monolayer cell membrane is detached from the culture dish, the PVDF membrane can be adopted to adsorb the monolayer cell membrane to the surface, and the monolayer cell membrane can be washed for several times by using physiological saline according to actual needs. Preferably, the PVDF membrane carries a hydrophilic modification matrix, and thus, the adsorption effect thereof to the monolayer cell thin film can be further improved. More preferably, the PVDF membrane is a star-poly (dimethylaminoethyl acrylate) polymer hydrophilically modified PVDF membrane. Therefore, the adsorption effect on the monolayer cell film is better.
In addition, according to an embodiment of the present invention, referring to fig. 3 and 4, the PVDF film described above includes a hollow region. Therefore, the surface of the membrane has certain gaps, and the operation of adsorbing the monolayer cell membrane is more convenient. In some embodiments of the invention, the PVDF membrane is circular (as shown in fig. 3).
Further, according to an embodiment of the present invention, the method for preparing a cell membrane may further include: and (3) superposing a plurality of monolayer cell films so as to obtain a composite cell film. Specifically, for example, 2 single-layer cell films are stacked, 2 single-layer cell films may be collected by using 2 PVDF films, and then the 2 PVDF films are bonded in a direction in which the cell film surfaces are opposite to each other, so as to obtain the stacked 2 single-layer cell films.
According to the embodiment of the invention, the prepared composite cell film can comprise 2-3 layers of cell films. Thereby, a better therapeutic effect can be obtained.
For ease of understanding, the above-described star-poly (dimethylaminoethyl acrylate) polymer hydrophilically-modified PVDF membrane is described in detail below.
The star-shaped poly (dimethylaminoethyl acrylate) (star-shaped PDMAEA) has a structure shown in a formula I
Figure BDA0002924945050000121
In the formula I, R is
Figure BDA0002924945050000122
n is a positive integer of 15 to 105. The polymerization degree of the star-poly (dimethylaminoethyl acrylate) polymer is between 100 and 400, and the molecular weight of the star-poly (dimethylaminoethyl acrylate) polymer is between 10,000 and 60,000 daltons. The inventor finds that the long alkyl chain in the star-poly (dimethylaminoethyl acrylate) polymer has hydrophobicity and good compatibility with PVDF; the dimethylaminoethyl acrylate chain segment has high hydrophilicity, the star structure also helps the star-polydimethylaminoethyl acrylate polymer to be firmly fixed on the matrix membrane, and the hydrophilicity is improved, and the stability of the combination of the modifier and the PVDF membrane is also improved. Therefore, the star-poly (dimethylaminoethyl acrylate) polymer hydrophilic modified PVDF membrane has higher hydrophilicity, permeability and stain resistance, and still has higher recovery performance after being used for a plurality of times and for a long time.
According to one embodiment of the present invention, a star-poly (dimethylaminoethyl acrylate) polymer is prepared as follows:
dissolving a proper amount of initiator azodiisobutyronitrile and star-shaped chain transfer reagent tetra-branched 2- (dodecyl trithiocarbonate) -2-methylpropanoic acid (the structure is shown in formula II) with the molar ratio of (60-420): 1 in 50mL of 2-butanone, removing oxygen in a reaction container by adopting a freezing-degassing-unfreezing method, injecting nitrogen or argon as protective gas, and placing in a metal bath at 70-120 ℃ for stirring reaction for 4-20 hours; dropping the obtained polymer solution into hexane, collecting the product by precipitation-precipitation method, removing impurities from the obtained product by rotary evaporation, vacuum drying and other steps, and drying to constant weight to obtain star-shaped poly (dimethylaminoethyl acrylate) polymer, wherein the nuclear magnetic resonance hydrogen spectrum of the star-shaped poly (dimethylaminoethyl acrylate) polymer is shown in figure 5, and the SEM spectrum is shown in figure 6.
Figure BDA0002924945050000131
Wherein R' is
Figure BDA0002924945050000132
And subsequently, adding the prepared star-poly (dimethylaminoethyl acrylate) polymer serving as an additive into a PVDF membrane casting solution, and preparing the hydrophilic modified PVDF membrane of the star-poly (dimethylaminoethyl acrylate) polymer by adopting a blade coating method. The specific method comprises the following steps: adding 50g of PVDF, 2g of PVP, 2.7g of star-shaped-poly (dimethylaminoethyl acrylate) polymer and a proper amount of DMF (dimethyl formamide) into a reaction vessel, stirring and reacting for 10-15 h in a metal bath at 70 ℃, and then degassing for 8h in a vacuum oven at 60 ℃ to obtain a membrane casting solution. The PVDF flat membrane is prepared by adopting a solvent induced phase separation method, water is used as a coagulating bath, the temperature is room temperature, the membrane casting solution is poured on a clean glass plate, an automatic membrane scraping machine is used for scraping the membrane, and the scraper thickness is 150 mu m. And (3) exposing the scraped membrane in air for 30s, putting the scraped membrane into a coagulating bath until the scraped membrane falls off on a glass plate, soaking the prepared membrane (M-0, M-1, M-2, M-3 and M-4) in distilled water, and changing water every 12h to remove residual solvent and pore-forming agent in the membrane to obtain a star-poly (dimethylaminoethyl acrylate) polymer hydrophilic modified PVDF membrane product.
The prepared product was subjected to a water contact angle test, and the results are shown in fig. 7. It can be seen that compared to the control sample with 0% star-polydimethylaminoethyl acrylate, PVDF membranes prepared with 5% and 10% star-polydimethylaminoethyl acrylate significantly decreased water contact angle and increased hydrophilicity.
In a fifth aspect of the invention, the invention provides a composite cell membrane. According to an embodiment of the present invention, the composite cell membrane includes: a plurality of monolayer cell membranes stacked, wherein cells of the monolayer cell membranes are treated with a calcium-magnesium free buffer. Specifically, the monolayer cell film is composed of a film made of a material having a self-assembly propertyEnergy of not more than 90mJ/m 2 Culturing in coated culture medium, and further treating with calcium-free magnesium buffer solution. Therefore, the preparation method of the composite cell film is simple, convenient and efficient, and has better treatment effect.
According to an embodiment of the present invention, the composite cell membrane includes 2-3 cell membranes. Wherein, the structure of the composite cell membrane comprising 3 layers of cell membranes is schematically shown in FIG. 8.
According to an embodiment of the present invention, the monolayer cell membrane is formed by the method for preparing a cell membrane of the above embodiment.
In a sixth aspect of the invention, the invention proposes the use of the composite cell membrane of the above embodiment in the preparation of a medicament. According to an embodiment of the invention, the medicament is for the treatment of at least one selected from the group consisting of: ocular injury repair and regeneration, skin and soft tissue necrosis, repair of wounded tissue, skin burn, cold injury, kidney transplantation, liver transplantation, heart transplantation, lung transplantation, soft tissue transplantation, wound repair, cosmetic surgery, abdominal hernia repair, liver injury, spleen rupture, stomach injury, duodenal injury, small intestine and mesenteric injury, gastric ulcer, duodenal ulcer, diabetic foot, central nervous system injury repair and regeneration, craniocerebral injury, spinal cord injury, myocarditis, myocardial infarction, atrial septal defect, ventricular septal defect, fracture healing, joint injury, or development of artificial bone. As described above, the method for preparing the composite cell membrane of the above embodiments is simple and efficient, and the therapeutic effect is better. Therefore, the composite cell film is used for preparing medicines, and a better treatment effect can be obtained.
In a seventh aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: the composite cell membrane of the above example; and pharmaceutically acceptable adjuvants. As described above, the method for preparing the composite cell membrane of the above embodiments is simple and efficient, and the therapeutic effect is better. Therefore, the composite cell membrane is used for the pharmaceutical composition, and a better treatment effect can be obtained.
It should be noted that the specific type of the above pharmaceutically acceptable excipients is not particularly limited, and those skilled in the art can select pharmaceutically acceptable excipients commonly used in the art according to actual needs.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Example 1
As shown in FIG. 9, the culture dish comprises a cell-bearing dish 1, an antiseptic cover 2, and a poly (2-vinylpyridine-co-styrene) block copolymer coating 3. The anti-bacterial cover 2 can be fastened to the cell-holding dish 1 with a certain gap between them for gas exchange with the outside.
The cell bearing dish 1 and the antibacterial cover 2 are cylinders, the diameter of the cell bearing dish 1 is 35mm, and the actual culture area is about 8.8cm 2 And the height is 13 mm. The cell bearing dish 1 and the antibacterial cover 2 are made of polystyrene materials, so that high light transmittance of the cell culture dish is ensured, and the cell morphology in vitro culture can be observed conveniently. The bottom of the cell-holding dish 1 had a coating 3 of poly (2-vinylpyridine-co-styrene) block copolymer with a thickness of 8nm, produced by vacuum evaporation.
Referring to FIG. 10, the culture dish was sufficiently sterilized by irradiation sterilization and then used for in vitro cell culture to prepare a cell membrane. In this example, mesenchymal stem cells within P5 were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine, and 1% non-essential amino acids at 37 ℃ in an atmosphere of 5% carbon dioxide. When the cells are cultured until the fusion degree reaches more than 90%, digesting the cells by trypsin, inoculating the cells on a culture dish at the density of 10,000 cells/culture dish, continuously culturing the cells by the same culture medium and conditions, removing the culture medium and washing a cell film by PBS when the cells grow and proliferate until the fusion degree reaches more than 95%, adding DPBS to completely cover the cell film, and then placing at room temperature until the cell film falls off by itself. 15min after replacing the medium with DPBS, the cell membrane completely detached from the cell culture dish and floated in the DPBS solution as a complete monolayer of membrane (see fig. 11 and 12). After several washes with physiological saline, the cell membranes can be used for further research or application.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
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, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (26)

1. A culture dish, comprising:
a body defining a culture space therein; and
a coating disposed at the bottom of the culture space, wherein the coating has a surface free energy of no more than 90mJ/m 2 。
2. A culture dish according to claim 1, wherein the thickness of the coating does not exceed 300 nm.
3. A culture dish according to claim 1, wherein the thickness of the coating is not less than 2nm, but not more than 100nm, preferably not more than 50nm, more preferably not more than 30nm, most preferably not more than 10 nm.
4. A culture dish according to claim 1, wherein the surface free energy of the coating does not exceed 60mJ/m 2 。
5. The culture dish of claim 1, wherein the coating comprises a polymer or block copolymer formed from at least one of the following monomers: vinylimidazole, vinylpyrrolidone, aminostyrene, methacrylamide, dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, cyanomethylstyrene, 1-methylpyridine chloride, vinylcaprolactam, acrylic acid, N-dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, propyl 3- (dimethylamino) acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, caprolactone, N-dimethylvinylbenzylamine, 4-vinylpyridine, divinylbenzene, vinyl benzoate, benzyl methacrylate, cyclohexyl methacrylate, butyl methacrylate, isopropyl methacrylate, acrylamide, allyl methacrylate, isocyanoethyl methacrylate, isopropyl methacrylate, methyl methacrylate, ethyl acrylate, methyl methacrylate, ethyl acrylate, and the like, Ethylene glycol dimethacrylate, ethylene glycol methyl methacrylate, polyhydroxyethyl methacrylate, 1,2, 4-triethylcyclohexane, furfuryl alcohol methyl ester, tetrahydrofurfuryl methacrylate, hexyl methacrylate, hydroxyethyl methacrylate, glycidyl methacrylate, propynyl methacrylate, 1,4-butanediol vinyl ether, isobornyl acrylate, ethylene glycol diacrylate, propynyl 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, dimethylglycidyl methacrylate, and dimethylglycidyl methacrylate, Heptafluorobutyl methacrylate, 1,1,1,3,3,3-hexafluoroisopropyl methacrylate, 2,3,3,4,4-hexafluoro-1, 5-pentasil-ylate, 2- (perfluorohexyl) ethyl methacrylate, trifluoroethyl methacrylate, pentafluorophenyl methacrylate, 1H,7H-dodecafluoroheptyl acrylate, 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-diethylaminoethyl acrylate, N-dimethylhexyl methacrylate, N-dimethyldecyl methacrylate, N-dimethylhexyl methacrylate, N-dimethyldecyl acrylate, N-dimethylhexyl methacrylate, N-dimethyloctyl methacrylate, N-dimethylhexyl methacrylate, N-methyl methacrylate, N-dimethylhexyl methacrylate, N-dodecylhexyl methacrylate, and N-dodecylhexyl methacrylate, Ethyl 2- (dimethylamino) acrylate, propyl 3- (dimethylamino) acrylate, ethyl 2- (dimethylamino) methacrylate, ethyl 2- (tert-butylamino) methacrylate, N-dimethylvinylbenzylamine, methacrylic acid, acrylamide, vinyl-N-methylpyridine chloride, N-dimethylvinylbenzylamine.
6. The culture dish of claim 5, wherein the coating is formed from a poly (2-vinylpyridine-co-styrene) block copolymer.
7. The culture dish of claim 5, wherein the coating is formed by at least one of a solution evaporation method, a spray coating method, a vacuum evaporation coating method, a chemical vapor deposition method, a pulsed laser deposition method, and a sol-gel method.
8. A culture dish according to claim 5, wherein the coating is formed by solid phase deposition of 2-vinylpyridine and styrene.
9. The culture dish of claim 1, further comprising:
a bottom plate detachably provided at the bottom of the culture space, wherein the coating layer is provided on a surface of the bottom plate.
10. Use of a culture dish according to any of claims 1 to 9 for the preparation of a thin film of cells.
11. A method of adjusting binding of cells to a culture dish according to any one of claims 1 to 8, the method comprising: the concentration of calcium and/or magnesium ions in the cell culture fluid is altered.
12. A method of preparing a thin film of cells, comprising:
culturing cells in a culture dish according to any one of claims 1 to 8 by using a liquid culture medium to form a monolayer cell membrane;
subjecting the monolayer cell film to a stripping solution having a lower concentration of cations than the liquid medium, the cations comprising at least one of calcium and/or magnesium ions; and
collecting the monolayer cell film from the stripping solution.
13. The method of claim 12, wherein the cells comprise at least one selected from stem cells, neuronal cells, astrocytes, oligodendrocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts;
optionally, the stem cells include at least one of induced pluripotent stem cells, adult stem cells, mesenchymal stem cells, neural stem cells, cardiac stem cells, and lung stem cells;
optionally, the mesenchymal stem cells comprise at least one of bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells;
optionally, the epithelial cells include at least one of corneal epithelial cells, prostate epithelial cells, renal tubular epithelial cells, coronary artery cells;
optionally, the endothelial cells comprise at least one of vascular endothelial cells, pulmonary artery endothelial cells, aortic endothelial cells;
optionally, the muscle cells comprise at least one of aortic smooth muscle cells, pulmonary smooth muscle cells, coronary smooth muscle cells;
optionally, the fibroblasts include at least one of cardiac fibroblasts, skin fibroblasts, renal interstitial fibroblasts, preferably mesenchymal stem cells.
14. The method according to claim 12, wherein the stripping solution is a buffer solution containing no calcium ions and no magnesium ions.
15. The method of claim 14, wherein the buffer has a pH of 6.9 to 7.4, and preferably the buffer is DPBS.
16. The method according to claim 12, wherein the collecting of the monolayer cell membrane is performed using a PVDF membrane.
17. The method according to claim 16, wherein the PVDF membrane carries hydrophilic modification groups, preferably wherein the PVDF membrane is a star-poly (dimethylaminoethyl acrylate) polymer hydrophilic modified PVDF membrane.
18. The method of claim 16 or 17, wherein the PVDF membrane comprises hollow regions.
19. The method of claim 18, wherein the PVDF membrane is circular.
20. The method of claim 12, further comprising:
and superposing a plurality of the monolayer cell films so as to obtain a composite cell film.
21. The method of claim 20, wherein the composite cell membrane comprises 2-3 layers of cell membranes.
22. A composite cell membrane, comprising:
a plurality of monolayer cell membranes stacked, wherein the cells of the monolayer cell membranes are treated with a calcium-magnesium free buffer.
23. The composite cell membrane of claim 22, wherein the composite cell membrane comprises 2-3 layers of cell membranes.
24. The composite cellular film according to claim 22, wherein the monolayer cellular film is formed by the method of any one of claims 12 to 21.
25. Use of a composite cell membrane as claimed in any one of claims 22 to 24 in the manufacture of a medicament for the treatment of at least one selected from the group consisting of:
ocular injury repair and regeneration, skin and soft tissue necrosis, repair of wounded tissue, skin burn, cold injury, kidney transplantation, liver transplantation, heart transplantation, lung transplantation, soft tissue transplantation, wound repair, cosmetic surgery, abdominal hernia repair, liver injury, spleen rupture, stomach injury, duodenal injury, small intestine and mesenteric injury, gastric ulcer, duodenal ulcer, diabetic foot, central nervous system injury repair and regeneration, craniocerebral injury, spinal cord injury, myocarditis, myocardial infarction, atrial septal defect, ventricular septal defect, fracture healing, joint injury, or development of artificial bone.
26. A pharmaceutical composition, comprising:
the composite cell membrane of any one of claims 22 to 24; and
pharmaceutically acceptable auxiliary materials.
CN202110129346.1A 2021-01-29 2021-01-29 Culture dish and application thereof in preparing cell film Pending CN114806869A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115414310A (en) * 2021-05-31 2022-12-02 深圳市碳源生物科技有限公司 Mesenchymal stem cell mask and preparation method thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115414310A (en) * 2021-05-31 2022-12-02 深圳市碳源生物科技有限公司 Mesenchymal stem cell mask and preparation method thereof
CN115414310B (en) * 2021-05-31 2023-10-31 深圳市碳源生物科技有限公司 Mesenchymal stem cell mask and preparation method thereof

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