Culture dishes and their use in preparing cell monolayers

Abstract

The application discloses a culture dish and its use in preparing a cell sheet. The culture dish comprises a body defining a culture space inside, and a coating arranged at the bottom of the culture space, wherein the surface free energy of the coating is not more than 90 mJ / m 2 . Thus, by adjusting the ion concentration in the cell liquid medium, the binding force between the coating and the cells can be reduced, so that the adherent cultured cells can be detached from the coating at the bottom of the culture dish in the form of a cell sheet, and the cell sheet is effectively obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular, the present application relates to the field of bioengineering, more particularly, the present application relates to a culture dish and its use in preparing a cell sheet, in particular, the present application relates to a culture dish, the use of a culture dish in preparing a cell sheet, a method for adjusting the binding force between cells and a culture dish, a method for preparing a cell sheet, a composite cell sheet, the use of a composite cell sheet in preparing a drug, and a pharmaceutical composition. BACKGROUND

[0002] With the development of biology and tissue engineering technology, biodegradable medical polymer implants and polymer-cell composite implants have attracted wide attention in the aspects of injury repair, tissue regeneration and organ transplantation. However, the immune response caused by biodegradable polymer materials and the inflammatory response caused by the decomposition of biodegradable polymer materials limit their clinical application; other methods also have obvious shortcomings, for example, after mixing the cell suspension and the polymer solution and infusion, the extracellular matrix (ECM) is often destroyed by hydrolase, which often causes low cell regeneration efficiency and utilization rate after reinfusion. In order to solve the above problems, cell sheet as a cell material without exogenous substances has attracted wide attention in cell transplantation applications.

[0003] The cell sheet refers to a complete monolayer cell sheet obtained by culturing isolated cells on a specific material under in vitro culture conditions, and separating the cells from the culture substrate without destroying the cell connection. Compared with cell injection and polymer-cell composite implants, the cell sheet has the following advantages in clinical application: (1) after traditional cell vein / artery injection, the distribution is biased towards liver, lung and other tissues, and it is difficult to realize the targeting of mesenchymal stem cells to the transplantation organ site; while the cell sheet can be directly transplanted to the required site during transplantation surgery. (2) Traditional cell injection system often has poor curative effect for some special parts of diseases, such as bone joints, tendons, etc., due to the lack of rich capillary network in the tissue; while the cell sheet can be directly applied to these parts to play a key therapeutic role. (3) The cell sheet does not need the support of biodegradable polymer materials, so it is not easy to cause the immune response and inflammatory response caused by polymer materials. (4) The cell sheet retains the extracellular matrix, which is beneficial to the regeneration and in vivo utilization of cells.

[0004] The current technique for preparing cell sheet is mainly based on the temperature-sensitive polymer non-adhesion cell culture vessel developed by Teruo Okano et al. However, this preparation method has significant disadvantages, such as the non-uniform change of the surface chemical properties of the culture vessel caused by temperature change, long time consumption for preparing cell sheet, and difficult preparation method, etc. Other preparation methods such as ultrasonic stimulation method, electric stimulation method, ultraviolet irradiation method, etc. also have disadvantages. Therefore, the current means for preparing cell sheet still needs to be improved. SUMMARY

[0005] The present application aims to at least partially solve one of the technical problems in the related art. To this end, one object of the present application is to provide a means capable of effectively obtaining cell sheet by adjusting the ion concentration to make the adherent cells detach from the culture vessel.

[0006] In a first aspect of the present application, a culture vessel is provided. According to embodiments of the present application, the culture vessel comprises a body defining a culture space therein, and a coating layer disposed at the bottom of the culture space, wherein the surface free energy of the coating layer is no more than 90 mJ / m 2 .

[0007] According to embodiments of the present application, when the culture vessel is used to culture cells such as stem cells, especially when liquid medium is used for culture, the cells can adhere to the coating layer at the bottom of the culture space during the expansion process and can proliferate adherently on the coating layer. In addition, since the surface free energy of the coating layer is no more than 90 mJ / m 2 , by adjusting the ion concentration in the cell liquid medium, the binding force between the coating layer and the cells can be reduced, so that the adherent cultured cells can be detached from the coating layer at the bottom of the culture vessel in the form of cell sheet, and the cell sheet can be effectively obtained. The inventors have unexpectedly found that the cell sheet can be quickly detached from the bottom of the culture vessel using this method, usually within 1 hour after changing the ion concentration, for example, within 30 minutes, such as within 20 minutes, for example, 15 minutes. The reason for this may be that by changing the ion concentration in the culture medium, such as the concentration of calcium ions and / or magnesium ions, the conformation of the cell focal adhesion complex transmembrane receptor will change, the binding force between the coating layer and the cells will decrease, so that the cells can be detached from the bottom surface of the culture vessel without trypsin hydrolysis of the intercellular protein, and a complete cell sheet can be obtained.

[0008] According to embodiments of the present application, the above-mentioned culture vessel can also have at least one of the following additional technical features:

[0009] According to embodiments of the present application, the thickness of the coating is not more than 300 nm. According to embodiments of the present application, the inventors found that if the thickness of the coating is more than 300 nm, the growth of the cells is adversely affected.

[0010] According to embodiments of the present application, the thickness of the coating is not less than 2 nm, but not more than 100 nm, preferably not more than 50 nm, more preferably not more than 30 nm, most preferably not more than 10 nm. Thereby, the effect of detachment of the cell sheet from the culture dish is better.

[0011] According to embodiments of the present application, the surface free energy of the coating is not more than 60 mJ / m 2 . Thereby, the effect of detachment of the cell sheet from the culture dish is better.

[0012] According to embodiments of the present application, the coating contains a high polymer or block copolymer formed from at least one of the following monomers: vinyl imidazole, vinyl pyrrolidone, aminostyrene, methacrylamide, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, hexalactone, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, hexalactone, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, hexalactone, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, hexalactone, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, hexalactone, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride, 2N-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 alcohol methyl ester methacrylate), tetrahydrofurfuryl methacrylate, hexylmethacrylate, hydroxyethyl methacrylate, glycidyl methacrylate, propargylmethacrylate, 1,4-butanediol divinylether, isobornyl acrylate, ethylene glycol diacrylate, propargyl acrylate, 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane, hexavinyldisiloxane, 2,4,6,6-trivinyl-2,4,6-trimethylcyclotrisiloxane5-trimethylcyclotrisiloxane, 2,4,6-trimethyl-2,4,6-trivinylcyclotrisilazane, dimethylphenylvinylsilane, heptadecafluorodecyl methacrylate, perfluorodecyl acrylate, heptafluorobutylmethacrylate, 1,1,1,3,3,3-hexafluoroisopropylmethacrylate, 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-acrylateN-diethylaminoethyl acrylate, 2-(dimethylamino)ethyl acrylate, 3-(dimethylamino)propyl acrylate, 2-(dimethylamino)ethyl methacrylate, t-butylaminoethyl methacrylate, N,N-dimethylaminomethyl styrene, methacrylic acid, acrylamide, vinyl-N-methylpyridinium chloride, N-(4-vinylbenzyl)-N,N-dimethylamine.

[0013] According to an embodiment of the present application, the coating layer is formed of a poly(2-vinylpyridine-co-styrene) block copolymer. Thereby, the effect of detaching the cell sheet from the culture dish is better.

[0014] According to an embodiment of the present application, the coating layer is formed by at least one of a solution evaporation method, a spray method, a vacuum evaporation plating method, a chemical vapor deposition method, a pulsed laser deposition method, and a sol-gel method.

[0015] According to an embodiment of the present application, the coating layer is formed by solid phase deposition of 2-vinylpyridine and styrene.

[0016] According to an embodiment of the present application, the culture dish further comprises a bottom plate detachably disposed at the bottom of the culture space, wherein the coating layer is disposed on the surface of the bottom plate.

[0017] In the second aspect of the present application, the present application provides a use of the culture dish of the above-mentioned embodiments in preparing a cell sheet. As mentioned above, the culture space of the culture dish has a coating layer with a free energy of no more than 90 mJ / m 2 Thereby, by adjusting the ion concentration in the cell liquid medium, the binding force between the coating layer and the cell can be reduced, so that the adherent cultured cell can be detached from the coating layer at the bottom of the culture dish in the form of a cell sheet, and the cell sheet can be effectively obtained.

[0018] In a third aspect, the present application provides a method for adjusting the binding force between cells and a culture dish. According to embodiments of the present application, the culture dish is the culture dish of the above embodiments, and the method comprises: changing the concentration of calcium ions and / or magnesium ions in the cell culture medium. The inventors have surprisingly found that by changing the concentration of ions in the culture medium, such as the concentration of calcium ions and / or magnesium ions, the configuration of the transmembrane receptor of the focal adhesion complex will change accordingly, and thus the binding force between the coating and the cells will decrease. Further, by this method, a cell sheet can be efficiently prepared.

[0019] In a fourth aspect, the present application provides a method for preparing a cell sheet. According to embodiments of the present application, the method comprises: culturing cells in the culture dish of the above embodiments using a liquid medium to form a monolayer cell sheet; placing the monolayer cell sheet in a stripping solution, wherein the concentration of cations in the stripping solution is lower than that in the liquid medium, and the cations include at least one of calcium ions and / or magnesium ions; and collecting the monolayer cell sheet from the stripping solution.

[0020] According to embodiments of the present application, when cells such as stem cells are cultured in the culture dish of the above embodiments, the cells can adhere to the coating on the bottom of the culture space during the expansion process and can proliferate on the coating. In addition, since the surface free energy of the coating in the culture dish is not more than 90 mJ / m 2 By adjusting the ion concentration in the culture medium by using a stripping solution with a lower concentration of calcium ions and / or magnesium ions than the liquid medium, the binding force between the coating and the cells can be reduced, allowing the adherent cells to detach from the coating on the bottom of the culture dish in the form of a cell sheet, effectively obtaining a cell sheet. The inventors have surprisingly found that this method can quickly complete the detachment of the cell sheet from the bottom of the culture dish, usually within 1 hour after changing the ion concentration, for example, within 30 minutes, such as within 20 minutes, for example, 15 minutes. The reason for this may be that by changing the concentration of calcium ions and / or magnesium ions in the culture medium, the configuration of the transmembrane receptor of the focal adhesion complex changes, the binding force between the coating and the cells decreases, and thus the cells can be detached from the bottom surface of the culture dish without trypsin hydrolysis of the intercellular proteins, obtaining a complete cell sheet.

[0021] According to embodiments of the present application, the method for preparing a cell sheet can further have at least one of the following additional technical features:

[0022] According to embodiments of the present application, the cells include at least one selected from stem cells, neuronal cells, astrocytes, oligodendrocytes, epithelial cells, endothelial cells, muscle cells, and fibroblasts.

[0023] According to an embodiment of the present application, the stem cells comprise at least one of induced pluripotent stem cells, adult stem cells, mesenchymal stem cells, neural stem cells, cardiac stem cells and lung stem cells.

[0024] According to an embodiment of the present application, the mesenchymal stem cells comprise at least one of bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells.

[0025] According to an embodiment of the present application, the epithelial cells comprise at least one of corneal epithelial cells, prostate epithelial cells, renal tubular epithelial cells, coronary artery epithelial cells.

[0026] According to an embodiment of the present application, the endothelial cells comprise at least one of arterial endothelial cells, pulmonary artery endothelial cells, aortic endothelial cells.

[0027] According to an embodiment of the present application, the muscle cells comprise at least one of aortic smooth muscle cells, pulmonary artery smooth muscle cells, coronary artery smooth muscle cells.

[0028] According to an embodiment of the present application, the fibroblasts comprise at least one of cardiac fibroblasts, skin fibroblasts, renal interstitial fibroblasts, preferably mesenchymal stem cells.

[0029] According to an embodiment of the present application, the stripping solution is a buffer solution free of calcium ions and magnesium ions.

[0030] According to an embodiment of the present application, the buffer solution has a pH of 6.9-7.4, and preferably the buffer solution is DPBS.

[0031] According to an embodiment of the present application, the monolayer cell film is collected using a PVDF membrane.

[0032] According to an embodiment of the present application, the PVDF membrane carries a hydrophilic modification group, and preferably the PVDF membrane is a star-shaped-polydimethylaminoethyl acrylate polymer hydrophilically modified PVDF membrane.

[0033] According to an embodiment of the present application, the PVDF membrane comprises a hollow region.

[0034] According to an embodiment of the present application, the PVDF membrane is in the shape of a circular ring.

[0035] According to an embodiment of the present application, the method further comprises: stacking a plurality of the monolayer cell films to obtain a composite cell film.

[0036] According to an embodiment of the present application, the composite cell film comprises 2-3 layers of cell films.

[0037] In a fifth aspect, the present application provides a composite cell sheet. According to an embodiment of the present application, the composite cell sheet comprises: a plurality of single-layer cell sheets stacked together, wherein the cells of the single-layer cell sheets are treated with calcium-magnesium-free buffer. Specifically, the single-layer cell sheet is formed by the method for forming a cell sheet according to any one of the above-mentioned embodiments, and has a free energy of no more than 90 mJ / m 2 The composite cell sheet is cultured in a coating medium and further treated with calcium-magnesium-free buffer. Thus, the method for forming a composite cell sheet according to the present application is simple and efficient, and has better therapeutic effect.

[0038] According to an embodiment of the present application, the composite cell sheet can further have at least one of the following additional technical features:

[0039] According to an embodiment of the present application, the composite cell sheet comprises 2-3 single-layer cell sheets.

[0040] According to an embodiment of the present application, the single-layer cell sheet is formed by the method for forming a cell sheet according to any one of the above-mentioned embodiments.

[0041] In a sixth aspect, the present application provides a use of the composite cell sheet according to any one of the above-mentioned embodiments in the preparation of a medicament. According to an embodiment of the present application, the medicament is used for treating at least one selected from the group consisting of: eye injury repair and regeneration, skin and soft tissue necrosis, repair of wounded tissue, skin burn, frostbite, kidney transplantation, liver transplantation, heart transplantation, lung transplantation, soft tissue transplantation, wound repair, cosmetic surgery, repair of abdominal hernia, liver injury, spleen rupture, gastric injury, duodenal injury, small intestine and mesentery 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, and development of artificial bone. As described above, the method for forming a composite cell sheet according to any one of the above-mentioned embodiments is simple and efficient, and has better therapeutic effect. Thus, the composite cell sheet is used in the preparation of a medicament, and better therapeutic effect can be achieved.

[0042] In a seventh aspect, the present application provides a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition comprises: the composite cell sheet according to any one of the above-mentioned embodiments; and a pharmaceutically acceptable excipient. As described above, the method for forming a composite cell sheet according to any one of the above-mentioned embodiments is simple and efficient, and has better therapeutic effect. Thus, the composite cell sheet is used in a pharmaceutical composition, and better therapeutic effect can be achieved.

[0043] Additional aspects and advantages of the present application will be given in part in the following description, become apparent from the following description, or be understood by those skilled in the art through the practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0044] The above and / or additional aspects and advantages of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the following drawings of which:

[0045] Figure 1 is a structural schematic diagram of a culture dish according to one embodiment of the present application;

[0046] Figure 2 is a structural schematic diagram of a culture dish according to another embodiment of the present application;

[0047] Figure 3 is a structural schematic diagram of a PVDF membrane according to one embodiment of the present application;

[0048] Figure 4 is a structural schematic diagram of a PVDF membrane according to another embodiment of the present application;

[0049] Figure 5 is a 1H NMR spectrum of a star-shaped-poly (dimethylamino ethyl acrylate) polymer according to one embodiment of the present application;

[0050] Figure 6 is a SEM spectrum of a star-shaped-poly (dimethylamino ethyl acrylate) polymer according to one embodiment of the present application;

[0051] Figure 7 is a water contact angle test diagram of a star-shaped-poly (dimethylamino ethyl acrylate) polymer hydrophilic modified PVDF membrane according to one embodiment of the present application;

[0052] Figure 8 is a structural schematic diagram of a composite cell film according to one embodiment of the present application;

[0053] Figure 9 is a structural schematic diagram of a culture dish in Example 1;

[0054] Figure 10 is a schematic diagram of a preparation and application method of a culture dish in Example 1;

[0055] Figure 11 is a schematic diagram of a principle of separating a cell film from a culture dish under the condition of changing calcium and magnesium ion concentrations in Example 1;

[0056] Figure 12 is an effect diagram of a cell film peeling off from a culture dish in Example 1.

[0057] Reference Signs:

[0058] 100: body;

[0059] 110: culture space;

[0060] 200: coating;

[0061] 120: Base plate;

[0062] 300: PVDF membrane;

[0063] 40: Monolayer cell membrane;

[0064] 1: Cell support dish;

[0065] 2: Antibacterial cap;

[0066] 3: Poly(2-vinylpyridine-co-styrene) block copolymer coating. Detailed Implementation

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In the present application, unless otherwise explicitly specified and limited, a first feature is "on" or "under" a second feature can mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature is "over", "above" and "on top of" the second feature can mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is horizontally higher than the second feature. The first feature is "under", "below" and "underneath" the second feature can mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is horizontally lower than the second feature.

[0071] In a first aspect of the present application, the present application provides a culture dish. With reference to Figure 1 According to embodiments of the present application, the culture dish comprises a body 100 and a coating layer 200. A culture space 110 is defined in the body 100, and the coating layer 200 is arranged at the bottom of the culture space 110. The surface free energy of the coating layer 200 is not more than 90 mJ / m 2 More preferably, the surface free energy of the coating layer 200 is not more than 60 mJ / m 2 In this way, the effect of cell sheet detachment from the culture dish is better.

[0072] 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 application, the overall shape of the culture dish is circular, the cell bearing surface is a smooth flat bottom, the height is 13-17 cm, the diameter is 35-100 mm, and the effective cell culture area is 8-57 cm 2 .

[0073] In addition, the specific material of the culture dish body is not particularly limited, and is preferably a polystyrene substrate. In this way, the culture dish body has good light transmittance, which facilitates observation of cell morphology. In some embodiments of the present application, the light transmittance of the culture dish body to visible light with a wavelength of 400-800 nm is more than 80%.

[0074] According to embodiments of the present application, the thickness of the coating layer 200 is not more than 300 nm. The inventors have unexpectedly found that a too large thickness of the coating layer 200 is not conducive to the growth of cells thereon. Further, the thickness of the coating layer 200 is not less than 2 nm, but not more than 100 nm, preferably not more than 50 nm, more preferably not more than 30 nm, and most preferably not more than 10 nm. In this way, the effect of cell sheet detachment from the culture dish is better.

[0075] According to embodiments of the present application, the coating 200 contains a high polymer or block copolymer formed from at least one of the following monomers: vinyl imidazole, vinyl pyrrolidone, aminostyrene, methacrylamide, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, hexalactone, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, hexalactone, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, hexalactone, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, hexalactone, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinyl chloride, 2-vinylpyridine, acrylonitrile, hexalactone, N,N-dimethylacrylamide, N-isopropylacrylamide, 4-vinylbenzyl chloride, vinyl benzyl cyanide, 1-methylpyridinium chloride, N-vinylcaprolactam, acrylic acid, dimethylaminoethyl acrylate, chloroethyl acrylate, cyanoethyl acrylate, 3-(dimethylamino)propyl acrylate, propylene, styrene, vinylN-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 alcohol methyl ester methacrylate), tetrahydrofurfuryl 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, 2,4,6,6-trivinyl-2,4,6-trimethylcyclotrisiloxane5-trimethylcyclotrisiloxane), 2,4,6-trimethyl-2,4,6-trivinylcyclotrisilazane, dimethylphenylvinylsilane, heptadecafluorodecyl methacrylate, perfluorodecyl acrylate, heptafluorobutylmethacrylate, 1,1,1,3,3,3-hexafluoroisopropyl isobutylacrylate, 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-acrylateN-diethylaminoethyl acrylate, 2-(diethylamino)ethyl acrylate, 3-(dimethylamino)propyl acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(t-butylamino)ethyl methacrylate, N,N-dimethylaminomethylstyrene, methacrylic acid, acrylamide, vinyl-N-methylpyridinium chloride, N-(4-vinylbenzyl)-N,N-dimethylamine.

[0076] Preferably, the coating layer 200 is formed of a poly(2-vinylpyridine-co-styrene) block copolymer and polyvinylbenzene, wherein the ratio of the poly(2-vinylpyridine-co-styrene) block copolymer and the polyvinylbenzene can be (50-100):1. More preferably, the coating layer 200 is entirely formed of a poly(2-vinylpyridine-co-styrene) block copolymer.

[0077] In addition, the formation method of the coating layer 200 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 application, the coating layer 200 can be formed by at least one of a solution evaporation method, a spray coating method, a vacuum evaporation plating method, a chemical vapor deposition method, a pulsed laser deposition method, and a sol-gel method. When the material of the coating layer 200 is selected as a poly(2-vinylpyridine-co-styrene) block copolymer, it is preferable to be formed by solid phase deposition of 2-vinylpyridine and styrene.

[0078] According to embodiments of the present application, with reference to Figure 2 The petri dish of the present application can further include a bottom plate 120. The bottom plate 120 is detachably disposed at the bottom of the culture space 110, wherein the coating layer 200 is disposed on the surface of the bottom plate 120. Thereby, the convenience of taking out the cell film formed on the surface of the coating layer 200 from the petri dish can be further improved.

[0079] In a second aspect of the present application, the present application provides a use of the culture dish of the above-mentioned embodiments in preparing a cell sheet. As mentioned above, the culture space of the culture dish has a coating layer with a free energy of no more than 90 mJ / m 2 . Thus, by adjusting the ion concentration in the cell liquid culture medium, the binding force between the coating layer and the cells can be reduced, so that the adherent cells can be detached from the coating layer of the culture dish in the form of a cell sheet, and the cell sheet can be effectively obtained.

[0080] In a third aspect of the present application, the present application provides a method for adjusting the binding force between cells and a culture dish. According to the embodiments of the present application, the culture dish is the culture dish of the above-mentioned embodiments, and the method comprises: changing the concentration of calcium ions and / or magnesium ions in the cell culture medium. The inventors have surprisingly found that by changing the ion concentration in the culture medium, such as the concentration of calcium ions and / or magnesium ions, the configuration of the focal adhesion complex transmembrane receptor of the cells will change, and thus the binding force between the coating layer and the cells will be reduced. Further, by this method, the cell sheet can be efficiently prepared.

[0081] In a fourth aspect of the present application, the present application provides a method for preparing a cell sheet. According to the embodiments of the present application, the method comprises: culturing cells in the culture dish of the above-mentioned embodiments using a liquid culture medium to form a monolayer cell sheet; placing the monolayer cell sheet in a stripping solution, the stripping solution having a lower concentration of cations than the liquid culture medium, the cations including at least one of calcium ions and / or magnesium ions; and collecting the monolayer cell sheet from the stripping solution.

[0082] The method for preparing a cell sheet according to the embodiments of the present application will be further described in detail below.

[0083] The method for preparing a cell sheet according to the embodiments of the present application is not particularly limited to a specific type of cells, and for example, can be at least one selected from the group consisting of stem cells, neuronal cells, astrocytes, oligodendrocytes, epithelial cells, endothelial cells, muscle cells, and fibroblasts. Specifically, 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; the mesenchymal stem cells include at least one of bone marrow-derived mesenchymal stem cells, adipose tissue-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 epithelial cells; the endothelial cells include at least one of arterial endothelial cells, pulmonary artery endothelial cells, and aortic endothelial cells; the muscle cells include at least one of aortic smooth muscle cells, pulmonary artery smooth muscle cells, and coronary artery smooth muscle cells; and the fibroblasts include at least one of cardiac fibroblasts, skin fibroblasts, and renal interstitial fibroblasts. Preferably, the above-mentioned cells are mesenchymal stem cells.

[0084] According to the embodiment of the present application, the stripping solution is a buffer solution without calcium ions and magnesium ions. In this way, the stripping solution can reduce the binding force between the cell membrane and the culture dish more effectively. More preferably, the buffer solution without calcium ions and magnesium ions has a pH of 6.9-7.4, for example, a commercially available DPBS buffer solution can be used.

[0085] According to the embodiment of the present application, the PVDF membrane can be used to collect the single-layer cell membrane. Specifically, after the single-layer cell membrane is separated from the culture dish, the PVDF membrane can be used to adsorb the single-layer cell membrane to the surface, and the single-layer cell membrane can be washed several times with normal saline according to the actual needs. Preferably, the PVDF membrane carries a hydrophilic modified substrate, thereby further improving the adsorption effect of the single-layer cell membrane. More preferably, the PVDF membrane is a star-shaped polydimethylaminoethyl acrylate polymer hydrophilic modified PVDF membrane. In this way, the adsorption effect of the single-layer cell membrane is better.

[0086] In addition, according to the embodiment of the present application, referring to Figure 3 and 4 , the PVDF membrane includes a hollow region. In this way, the membrane surface has a certain gap, and the operation of adsorbing the single-layer cell membrane is more convenient. In some embodiments of the present application, the PVDF membrane is in the form of a circular ring (as shown in Figure 3 ).

[0087] Further, according to the embodiment of the present application, the method for preparing the cell membrane can further include: stacking a plurality of single-layer cell membranes to obtain a composite cell membrane. Specifically, taking the example of stacking two single-layer cell membranes, two PVDF membranes can be used to collect two single-layer cell membranes respectively, and then the two PVDF membranes are attached in the direction opposite to the cell membrane surface to obtain two stacked single-layer cell membranes.

[0088] According to the embodiment of the present application, the prepared composite cell membrane can include 2-3 layers of cell membranes. In this way, a better treatment effect can be obtained.

[0089] For the convenience of understanding, the star-shaped polydimethylaminoethyl acrylate polymer hydrophilic modified PVDF membrane is described in detail below.

[0090] The star-shaped polydimethylaminoethyl acrylate polymer (star-shaped PDMAEA) has a structure as shown in formula I

[0091]

[0092] In formula I, R is n is a positive integer from 15 to 105. The star-shaped polydimethylaminoethyl acrylate polymer has a polymerization degree of 100 to 400 and a molecular weight of 10,000 to 60,000 Dalton. The inventor finds that the long alkyl chain in the star-shaped polydimethylaminoethyl acrylate polymer has hydrophobicity and has good compatibility with PVDF; the dimethylaminoethyl acrylate segment has high hydrophilicity, and the star-shaped structure also helps the star-shaped polydimethylaminoethyl acrylate polymer to be more firmly fixed on the substrate film, thereby improving the hydrophilicity and increasing the stability of the modifier combined with the PVDF film. Thus, the PVDF film modified by the star-shaped polydimethylaminoethyl acrylate polymer has higher hydrophilicity, permeability and antifouling property, and still has high recovery performance after multiple and long-time use.

[0093] According to a specific embodiment of the present application, the preparation method of the star-shaped polydimethylaminoethyl acrylate polymer is as follows:

[0094] An appropriate amount of initiator azobisisobutyronitrile, dimethylaminoethyl acrylate and star-shaped chain transfer agent four-branched 2-(dodecyltrithiocarbonate)-2-methylpropionic acid (structure as shown in formula II) with a molar ratio of (60-420): 1 are dissolved in 50 mL 2-butanone, the oxygen in the reaction container is removed by freeze-degassing-freezing method, and nitrogen or argon is injected as protective gas, and then placed in a metal bath at 70-120℃ for stirring reaction for 4-20h; the obtained polymer solution is dropped into hexane, and the product is collected by precipitation method, and the obtained product is impurity-removed and dried to constant weight through steps of rotary evaporation and vacuum drying, to obtain the star-shaped polydimethylaminoethyl acrylate polymer, whose 1H NMR spectrum is as shown in Figure 5 , and SEM spectrum is as shown in Figure 6 .

[0095] wherein, R' is

[0096] Subsequently, the prepared star-poly (dimethylamino ethyl acrylate) polymer is added as an additive into a PVDF casting solution, and a star-poly (dimethylamino ethyl acrylate) polymer hydrophilic modified PVDF membrane is prepared by using a doctor blade method. The specific method comprises the following steps: 50 g of PVDF, 2 g of PVP, 2.7 g of star-poly (dimethylamino ethyl acrylate) polymer and an appropriate amount of DMF are added into a reaction container, and stirring reaction is carried out at 70°C in a metal bath for 10-15 h, and then the reaction container is placed in a vacuum oven at 60°C for 8 h of degassing, so as to obtain a casting solution. A PVDF flat membrane is prepared by using a solvent induced phase separation method, water is used as a coagulation bath, the temperature is room temperature, the casting solution is poured on a clean glass plate, and a doctor blade is used for casting, and the thickness of the doctor blade is 150 μm. After the cast membrane is exposed in air for 30 s, it is placed in the coagulation bath until it falls off the glass plate, and the prepared membrane (M-0, M-1, M-2, M-3 and M-4) is placed in distilled water, and the water is changed every 12 h, so as to remove the residual solvent and pore-forming agent in the membrane, and a star-poly (dimethylamino ethyl acrylate) polymer hydrophilic modified PVDF membrane product is obtained.

[0097] The prepared product is subjected to water contact angle test, and the results are shown in Figure 7 It can be seen that, compared with a control sample with 0% star-poly (dimethylamino ethyl acrylate) polymer, the water contact angle of the PVDF membrane prepared by adding 5% and 10% star-poly (dimethylamino ethyl acrylate) polymer is obviously reduced, and the hydrophilicity is obviously improved.

[0098] In a fifth aspect, the present application provides a composite cell membrane. According to an embodiment of the present application, the composite cell membrane comprises: a plurality of single-layer cell membranes stacked together, wherein the cells of the single-layer cell membranes are treated by a calcium-magnesium-free buffer. Specifically, the single-layer cell membranes are formed by the method for preparing a cell membrane according to the above embodiment, and the cells of the single-layer cell membranes have a free energy of no more than 90 mJ / m 2 The cells are cultured in a culture medium and further treated by a calcium-magnesium-free buffer. Thus, the method for preparing the composite cell membrane according to the present application is simple and efficient, and the treatment effect is better.

[0099] According to an embodiment of the present application, the composite cell membrane comprises 2-3 layers of cell membranes. The structure of the composite cell membrane comprising 3 layers of cell membranes is shown in Figure 8 .

[0100] According to an embodiment of the present application, the single-layer cell membrane is formed by the method for preparing a cell membrane according to the above embodiment.

[0101] In a sixth aspect, the present invention proposes the use of the composite cell membrane of the above embodiments in the preparation of a medicament. According to embodiments of the present invention, the medicament is used to treat at least one of the following: eye injury repair and regeneration, skin and soft tissue necrosis, wound tissue repair, skin burns, frostbite, 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 mesentery 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 the development of artificial bone. As described above, the preparation method of the composite cell membrane of the above embodiments is simple and efficient, and the therapeutic effect is better. Therefore, using this composite cell membrane to prepare a medicament can achieve better therapeutic effects.

[0102] In a seventh aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, the pharmaceutical composition comprises: the composite cell membrane described above; and pharmaceutically acceptable excipients. As previously stated, the preparation method of the composite cell membrane described above is simple, efficient, and yields better therapeutic effects. Therefore, using this composite cell membrane in a pharmaceutical composition can achieve even better therapeutic effects.

[0103] It should be noted that the specific types of pharmaceutically acceptable excipients mentioned above are not particularly limited, and those skilled in the art can select commonly used pharmaceutically acceptable excipients according to actual needs.

[0104] 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.

[0105] Example 1

[0106] like Figure 9 As shown, the culture dish includes a cell support dish 1, an antibacterial cap 2, and a poly(2-vinylpyridine-co-styrene) block copolymer coating 3. The antibacterial cap 2 can be attached to the cell support dish 1, with a certain gap between them to allow for gas exchange with the outside.

[0107] The cell culture dish 1 and the antibacterial cap 2 are cylindrical. The diameter of the cell culture dish 1 is 35 mm, and the actual culture area is approximately 8.8 cm². 2 The height is 13mm. The cell culture dish 1 and the antibacterial cap 2 are made of polystyrene material, which ensures the high light transmittance of the cell culture dish so as to observe the cell morphology during in vitro culture. The bottom of the cell culture dish 1 has a poly(2-vinylpyridine-co-styrene) block copolymer coating 3 with a thickness of 8nm prepared by vacuum evaporation.

[0108] Reference Figure 10 After the culture dish is sterilized by irradiation, it is used for in vitro cell culture to prepare a cell sheet. In this embodiment, mesenchymal stem cells are used as an example. The mesenchymal stem cells are cultured in a DMEM medium containing 10% fetal bovine serum, 1% penicillin / streptomycin, 1% glutamine, and 1% non-essential amino acids at 37°C in a 5% carbon dioxide environment. When the cells are cultured to a confluence of more than 90%, the cells are digested with trypsin and inoculated on a culture dish at a density of 10,000 cells / dish. The cells are continuously cultured in the same medium and under the same conditions. When the cells grow and proliferate to a confluence of more than 95%, the medium is removed and the cell sheet is washed with PBS. DPBS is added to completely cover the cell sheet, and the cell sheet is left at room temperature until it is spontaneously detached. After 15 minutes, the cell sheet is completely detached from the culture dish and floats in the DPBS solution in the form of a complete monolayer sheet (see Figure 11 and Figure 12 ). The cell sheet is washed several times with normal saline and can be used for further research or application.

[0109] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and modified by those skilled in the art without contradiction.

[0110] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A method of preparing a cell sheet, characterized by, The method comprises: culturing cells in a petri dish with a liquid medium to form a monolayer cell sheet; immersing the monolayer cell sheet in a stripping solution, the stripping solution having a lower concentration of cations than the liquid medium, the cations including at least one of calcium ions and magnesium ions; and collecting the monolayer cell sheet from the stripping solution, the PVDF membrane is a star-shaped polyvinylidene fluoride-polyacrylamide dimethylaminoethyl hydrophilic modified PVDF membrane. The petri dish comprises: a body defining a culture space inside; and a coating disposed on the bottom of the culture space, wherein the surface free energy of the coating is no more than 90 mJ / m 2 ; the coating is formed by a poly(2-vinylpyridine-co-styrene) block copolymer.

2. The method of claim 1, wherein, The thickness of the coating is not more than 300 nm.

3. The method of claim 1, wherein, The thickness of the coating is not less than 2 nm but not more than 100 nm.

4. The method of claim 3, wherein, The thickness of the coating is not more than 50 nm.

5. The method of claim 3, wherein, The thickness of the coating is not more than 30 nm.

6. The method of claim 3, wherein, The thickness of the coating is not more than 10 nm.

7. The method of claim 1, wherein, The surface free energy of the coating is not more than 60 mJ / m 2 .

8. The method of claim 1, wherein, The coating is formed by at least one of a solution evaporation method, a spray method, a vacuum evaporation plating method, a chemical vapor deposition method, a pulsed laser deposition method, and a sol-gel method.

9. The method of claim 1, wherein, The coating is formed by solid-phase deposition of 2-vinylpyridine and styrene.

10. The method of claim 1, wherein, The petri dish further comprises: a bottom plate detachably arranged at the bottom of the culture space, wherein the coating is arranged on the surface of the bottom plate.

11. The method of claim 1, wherein, The cells include at least one selected from stem cells, neuronal cells, astrocytes, oligodendrocytes, epithelial cells, endothelial cells, muscle cells, and fibroblasts.

12. The method of claim 11, wherein, 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.

13. The method of claim 12, wherein, The mesenchymal stem cells include at least one of bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells.

14. The method of claim 11, wherein, The epithelial cells include at least one of corneal epithelial cells, prostate epithelial cells, renal tubular epithelial cells, and coronary artery endothelial cells.

15. The method of claim 11, wherein, The endothelial cells include at least one of arterial endothelial cells, pulmonary artery endothelial cells, and aortic endothelial cells.

16. The method of claim 11, wherein, The muscle cells include at least one of aortic smooth muscle cells, pulmonary artery smooth muscle cells, and coronary artery smooth muscle cells.

17. The method of claim 11, wherein, The fibroblasts include at least one of myocardial fibroblasts, skin fibroblasts, and renal interstitial fibroblasts.

18. The method of claim 1, wherein, The stripping solution is a calcium ion and magnesium ion-free buffer.

19. The method of claim 18, wherein, The buffer has a pH of 6.9-7.

4.

20. The method of claim 18, wherein, The buffer is DPBS.

21. The method of claim 1, wherein, The PVDF membrane includes a hollow region.

22. The method of claim 21, wherein, The PVDF membrane is in the shape of a circular ring.

23. The method of claim 1, wherein, Further comprising: stacking a plurality of the monolayer cell sheets to obtain a composite cell sheet.

24. The method of claim 23, wherein, The composite cell sheet includes 2-3 cell sheets.

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