Coating liquid for ultralow-adsorption cell culture surface, treatment method and culture material

Abstract

The invention relates to the field of biological functions, in particular to an ultra-low adsorption cell culture surface coating liquid, a treatment method and a culture material. The invention provides a coating solution for ultralow adsorption of a cell culture surface. The coating solution is prepared from the following raw materials in parts by weight: 0.5 to 3 parts of a coating polymer A, 0.25 to 1.5 parts of a coating polymer B, 0.025 to 0.15 part of a light curing agent and 95.35 to 99.2 parts of water, the coating polymer A comprises a povidone polymer; the coating polymer B comprises a polyester polymer with a polyether chain segment; research finds that the coating polymer A and the coating polymer B can be directly cross-linked on the culture surface to form a polymer coating, and the formed polymer coating is promoted to be cured by a light curing agent, that is, through cooperation of the components, the ultralow-adsorption cell culture surface can be obtained; further, the cells can be promoted to stably form quasi-spheroids with uniform sizes after being cultured.

Description

Technical Field

[0001] The invention relates to the field of biological functions, and in particular to a coating solution and a treatment method for an ultra-low adsorption cell culture surface, and a culture material. Background Art

[0002] Traditional 2D monolayer cell cultures can hardly accurately reflect the in vivo growth environment of cells, which can easily lead to the loss of cell structure and tissue function. 3D cell culture technology can not only better simulate the natural environment of cell survival in organisms, but its natural conditions can also maintain cell-to-cell interactions and more realistic biochemical and physiological reactions.

[0003] 3D cell spheroids and organoids provide an attractive and biologically relevant 3D cell model system for current drug development, disease modeling, tumor detection and stem cell research. 3D tumor cell spheroids can mimic the uniform growth of solid tumors in vivo and the hypoxic core of large tumors, and are an effective model for detecting malignant cells and tumors; neural stem cells aggregate with each other through endogenously secreted natural extracellular matrix, leading to the rapid formation of neurospheres. The consistent cell growth in neurospheres can further differentiate into multicellular structures called brain organoids with tissue-specific morphology and function; embryonic stem cells and induced pluripotent stem cells form embryoid bodies, which are held together by a complex network of adhesion molecules. Paracrine signals inside embryoid bodies are very important for the formation of embryos. This signal can respond to environmental induction and determine the final differentiation of the three germ layers.

[0004] In these application areas, the induction process of cell spheroids on the culture surface is crucial. Such a culture surface should be able to inhibit nonspecific interactions between cells and the surface, eliminate unnecessary cell attachment, and ultimately allow users to culture cell spheroids of uniform size more conveniently and stably.

[0005] Currently, techniques for generating spheroids may include the use of reconstituted basement membranes, agarose, or hanging drop methods. These techniques can be time-consuming, may even require the purchase of additional equipment, and cannot guarantee the stability and homogeneity of spheroids. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention lies in a coating solution, a treatment method and a culture material for an ultra-low adsorption cell culture surface. By means of the coating solution, the treatment method and the culture material for an ultra-low adsorption cell culture surface, the adsorption performance of the culture material surface can be made ultra-low, thereby promoting the stable formation of pseudo-spheroids of uniform size after cell culture.

[0007] To this end, the present invention provides the following technical solutions:

[0008] A coating solution for ultra-low adsorption cell culture surface, comprising the following raw materials in parts by weight:

[0009] Coating polymer A 0.5-3 parts, coating polymer B 0.25-1.5 parts, light curing agent 0.025-0.15 parts, water 95.35-99.2 parts;

[0010] The coating polymer A comprises a polyvidone polymer;

[0011] The coating polymer B includes a polyester polymer having a polyether segment.

[0012] Optionally, the polyvidone polymer includes at least one of polystyrene diallyl acrylate, polyvinyl alcohol, polyvinyl pyrrolidone K30 and polyvinyl pyrrolidone K90.

[0013] Optionally, the polyester polymer having a polyether segment includes at least one of polyethylene glycol acrylate, polyethylene glycol diacrylate and poly(ethylene glycol) methyl ether methacrylate.

[0014] Optionally, the photocuring agent includes at least one of Irgacure 369, I2959 and ITX.

[0015] A method for treating an ultra-low adsorption cell culture surface comprises the following steps:

[0016] S1. Performing low-temperature plasma treatment on the surface of the material to be treated;

[0017] S2. Coating the surface of the material to be treated with the coating solution of the ultra-low adsorption cell culture surface;

[0018] S3, UV curing the surface of the material to be treated coated in step S2.

[0019] Optionally, in step S1, the low-temperature plasma is at least one of oxygen, nitrogen and argon;

[0020] And / or, the low temperature plasma treatment conditions are: treatment power is 500W-2000W, treatment time is 200s-2000s;

[0021] And / or, the material to be treated includes one of polystyrene, polypropylene, polycarbonate and polyethylene terephthalate.

[0022] Optionally, in step S2, the thickness of the coating solution of the ultra-low adsorption cell culture surface coating the surface of the material to be treated is 200-500 μm.

[0023] Optionally, in step S2, the coating liquid of the ultra-low adsorption cell culture surface is coated on the surface of the material to be treated by spray coating, immersion coating or spin coating; preferably, the spray coating includes atomization spraying, high-pressure spraying or electrostatic spraying.

[0024] Optionally, the UV curing includes using an ultraviolet lamp to irradiate the surface of the material to be treated coated in step S2, the wavelength of the ultraviolet lamp is 254nm or 365nm, and the irradiation time is 10min-60min.

[0025] An ultra-low adsorption cell culture material is obtained by the ultra-low adsorption cell culture surface treatment method.

[0026] As an practicable manner, the cells include but are not limited to tumor cells, neuronal cells and bone marrow mesenchymal stem cells.

[0027] The technical solution of the present invention has the following advantages:

[0028] 1. The present invention provides a coating solution for an ultra-low adsorption cell culture surface, comprising the following raw materials in parts by weight: 0.5-3 parts of a coating polymer A, 0.25-1.5 parts of a coating polymer B, 0.025-0.15 parts of a photocuring agent, and 95.35-99.2 parts of water; the coating polymer A comprises a polyvidone polymer; the coating polymer B comprises a polyester polymer having a polyether segment; the present invention has found that the above-mentioned coating polymer A and coating polymer B can be directly cross-linked on the culture surface to form a polymer coating, and the photocuring agent promotes the curing of the formed polymer coating under the irradiation of an ultraviolet lamp of a specific wavelength, that is, through the coordination of the above-mentioned components, an ultra-low adsorption cell culture surface can be obtained, and further, the cells can be promoted to stably form pseudo-spheroids of uniform size after culture.

[0029] 2. The present invention provides a coating solution for an ultra-low adsorption cell culture surface, wherein the polyvidone polymer includes at least one of polystyrene diallyl acrylate, polyvinyl alcohol, polyvinyl pyrrolidone K30 and polyvinyl pyrrolidone K90; the polyester polymer with a polyether segment includes at least one of polyethylene glycol acrylate, polyethylene glycol diacrylate and poly(ethylene glycol) methyl ether methacrylate; the present invention has found that the combination of the above-mentioned preferred polyester polymer with a polyether segment and a povidone polymer can further reduce the adsorption of the culture surface, thereby promoting the stable formation of uniform-sized pseudo-spheroids after cell culture.

[0030] 3. A method for treating an ultra-low adsorption cell culture surface, comprising the following steps: S1, subjecting the surface of a material to be treated to low-temperature plasma treatment; S2, coating the surface of the material to be treated with the coating solution of the ultra-low adsorption cell culture surface; S3, subjecting the coated surface of the material to be treated in step S2 to ultraviolet curing; in the above steps, in step S1, low-temperature plasma treatment, on the one hand, forms double bonds on the surface of the material to facilitate grafting of a polymer coating, and on the other hand, reduces the contact angle of the material surface, which is beneficial to the uniform distribution of the spraying liquid and can ensure that the coating liquid is successfully coated on the surface of the material to be treated; in step S2, during the coating process, using the coating solution with the formula and mass ratio can greatly reduce the surface adsorption performance; in step S1, during the ultraviolet curing process, the stability of the bonding force between the surface of the material to be treated and the coating can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 These are the observation results of cells cultured in 96-well plates in each group in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0033] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0034] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0035] The Chinese name of PVPP is polystyrene diallyl acrylate;

[0036] The Chinese name of PVA is polyvinyl alcohol;

[0037] The Chinese name of PVPK30 is polyvinyl pyrrolidone K30;

[0038] PVPK90 Chinese name is polyvinyl pyrrolidone K90

[0039] The Chinese name of PEGA is polyethylene glycol acrylate;

[0040] The Chinese name of PEGDA is polyethylene glycol diacrylate;

[0041] The Chinese name of PEGMA is poly(ethylene glycol) methyl ether methacrylate.

[0042] Irgacure 369, I2959 and ITX were purchased from SigmaAldrich.

[0043] The atomizing spray equipment used in the following examples was purchased from SIANSONIC, with a spraying power of 0.5-5 W, a flow rate of 0.1-0.8 ml / min, and a pressure of 0.1-3 psi.

[0044] Example

[0045] This embodiment 1-7 provides a coating solution for an ultra-low adsorption cell culture surface, the formula of which is as follows:

[0046] Table 1. Coating solution formula

[0047]

[0048]

[0049] The method for preparing the coating solution for the ultra-low adsorption cell culture surface in the above embodiment is to mix the raw materials according to the formula, stir at 50° C. for 12 hours, stir to dissolve, filter, and prepare the solution.

[0050] Example 8

[0051] This embodiment provides a method for treating an ultra-low adsorption cell culture surface, wherein the coating solution is the formula of embodiment 1, and comprises the following steps:

[0052] (1) The surface of the disposable consumables culture material is treated with low-temperature plasma. The material type of the disposable consumables is polystyrene, the gas for plasma treatment is oxygen, the treatment power is 1000 W, and the treatment time is 1000 s.

[0053] (2) The coating solution in Example 1 was used to evenly spray the coating solution on the culture surface after plasma treatment using an atomizing spraying device. The power and flow rate of the atomizing spraying device were adjusted to achieve a spraying thickness of 300 μm.

[0054] (3) Use ultraviolet light to irradiate the culture surface coated in step (2), the wavelength of the ultraviolet light is 365nm, and the irradiation time is 30min.

[0055] Example 9

[0056] This embodiment provides a method for treating an ultra-low adsorption cell culture surface, wherein the coating solution is the formula of embodiment 2, and comprises the following steps:

[0057] (1) First, the surface of the disposable consumables culture is treated with low-temperature plasma. The material type of the disposable consumables is polystyrene, the gas for plasma treatment is oxygen, the treatment power is 500 W, and the treatment time is 200 s.

[0058] (2) The coating solution of Example 2 was used to evenly spray the coating solution on the culture surface after plasma treatment using an atomizing spraying device. The power and flow rate of the atomizing spraying device were adjusted to achieve a spraying thickness of 200 μm.

[0059] (3) Use ultraviolet light to irradiate the coated culture surface. The wavelength of the ultraviolet light is 254 nm and the irradiation time is 10 min.

[0060] Example 10

[0061] This embodiment provides a method for treating an ultra-low adsorption cell culture surface, wherein the coating solution is the formulation of embodiment 3, and comprises the following steps:

[0062] (1) First, the surface of the disposable consumable culture material is treated with low-temperature plasma. The material type of the disposable consumable is polystyrene, the gas for plasma treatment is oxygen, the treatment power is 2000W, and the treatment time is 2000s.

[0063] (2) The coating solution of Example 3 was used to evenly spray the coating solution on the culture surface after the plasma treatment using an atomizing spraying device. The power and flow rate of the atomizing spraying device were adjusted to achieve a spraying thickness of 500 μm.

[0064] (3) Use ultraviolet light to irradiate the coated culture surface. The wavelength of the ultraviolet light is 365nm and the irradiation time is 60min.

[0065] Embodiment 11

[0066] The embodiment provides a method for treating an ultra-low adsorption cell culture surface, which is different from the embodiment 8 in that the coating solution used is the coating solution of the embodiment 4.

[0067] Example 12

[0068] The embodiment provides a method for treating an ultra-low adsorption cell culture surface, which is different from the embodiment 8 in that the coating solution used is the coating solution of the embodiment 5.

[0069] Embodiment 13

[0070] The embodiment provides a method for treating an ultra-low adsorption cell culture surface, which is different from the embodiment 8 in that the coating solution used is the coating solution of the embodiment 6.

[0071] Embodiment 14

[0072] The embodiment provides a method for treating an ultra-low adsorption cell culture surface, which is different from the embodiment 8 in that the coating solution used is the coating solution of the embodiment 7.

[0073] Comparative Example 1

[0074] This comparative example provides a coating solution, the formula of which is:

[0075] PEG600 (polyethylene glycol 600) 3g, deionized water 97g.

[0076] The preparation method of the coating solution is as follows: the raw materials are mixed according to the formula, stirred at 50° C. for 12 hours to dissolve, and filtered to obtain the coating solution.

[0077] Comparative Example 2

[0078] This comparative example provides a method for treating an ultra-low adsorption cell culture surface, using the coating solution of comparative example 1, comprising the following steps:

[0079] (1) First, the surface of the disposable consumable culture material is treated with low-temperature plasma. The material type of the disposable consumable is polystyrene, the gas for plasma treatment is oxygen, the treatment power is 2000W, and the treatment time is 2000s.

[0080] (2) The coating solution of Comparative Example 1 was used to evenly spray the coating solution on the culture surface after plasma treatment using an atomizing spraying device. The power and flow rate of the atomizing spraying device were adjusted to achieve a spraying thickness of 300 μm.

[0081] (3) Use ultraviolet light to irradiate the coated culture surface. The wavelength of the ultraviolet light is 365nm and the irradiation time is 60min.

[0082] Experimental Example 1

[0083] A 96-well plate (U bottom) was produced by the process parameters of Examples 8-14 and Comparative Example 2. Human breast cancer cells (MCF-7 cells) were selected, and a DMEM growth medium (with double antibodies) containing 10% FBS was used. A certain volume of the cell suspension was taken, and the cell suspension was diluted according to a certain ratio to prepare a cell density of 2.5×10 4Then, the prepared cell suspension was inoculated into each test well of a 96-well U-bottom cell culture plate at 0.2 mL / well, and observed after 7 days of culture. The 96-well plates of each embodiment and comparative example were observed under a Leica stereo microscope. The results are as follows: Figure 1 As shown, the cells in the 96-well plates of Examples 8 and 13 formed complete spheres, with the best effect. The cells in the 96-well plates of Examples 12 and 14 also almost formed complete spheres. The cells in the 96-well plates of Examples 9, 10, and 11 also aggregated into clusters, with almost no cells adhering to the wells, while the cells in the 96-well plate of Comparative Example 2 grew in large numbers by adhering to the wall.

[0084] Experimental Example 2

[0085] In Experimental Example 1, after 7 days of culture, the average particle size of each cell sphere (cell pseudo-spheroid) in the 96-well plate of each Example 8-14 and Comparative Example 2 was measured, that is, (the longest diameter + the shortest diameter) / 2, and the average particle size of the total cell spheres in each group and the coefficient of variation CV=σ / μ×100% were measured, σ is the standard deviation of the data, μ is the average value of the data, and it can be concluded that the CV value of the particle size of the cell spheres in each group is less than 10%, indicating that each group of cells stably forms pseudo-spheroids of uniform size after culture, among which Examples 8 and 13 are the best, with more uniform size.

[0086] Table 2. Average particle size of cell spheres in each group

[0087]

[0088]

[0089] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A coating solution for ultra-low adsorption cell culture surface, characterized in that: The invention comprises the following raw materials in parts by weight: Coating polymer A 0.5-3 parts, coating polymer B 0.25-1.5 parts, light curing agent 0.025-0.15 parts, water 95.35-99.2 parts; The coating polymer A comprises a polyvidone polymer; The coating polymer B includes a polyester polymer having a polyether segment.

2. The coating solution for ultra-low adsorption cell culture surface according to claim 1, characterized in that: The polyvidone polymer includes at least one of polystyrene diallyl acrylate, polyvinyl alcohol, polyvinyl pyrrolidone K30 and polyvinyl pyrrolidone K90.

3. The coating solution for ultra-low adsorption cell culture surface according to claim 1 or 2, characterized in that: The polyester polymer having a polyether segment includes at least one of polyethylene glycol acrylate, polyethylene glycol diacrylate and poly(ethylene glycol) methyl ether methacrylate.

4. The coating solution for ultra-low adsorption cell culture surface according to any one of claims 1 to 3, characterized in that: The photocuring agent includes at least one of Irgacure 369, I2959 and ITX.

5. A method for treating an ultra-low adsorption cell culture surface, characterized in that: The steps include: S1. Performing low-temperature plasma treatment on the surface of the material to be treated; S2. Coating the surface of the material to be treated with the coating solution of the ultra-low adsorption cell culture surface according to any one of claims 1 to 4; S3, UV curing the surface of the material to be treated coated in step S2.

6. The method for treating an ultra-low adsorption cell culture surface according to claim 5, characterized in that: In step S1, the low temperature plasma is at least one of oxygen, nitrogen and argon; And / or, the low temperature plasma treatment conditions are: treatment power is 500W-2000W, treatment time is 200s-2000s; And / or, the material to be treated includes one of polystyrene, polypropylene, polycarbonate and polyethylene terephthalate.

7. The method for treating an ultra-low adsorption cell culture surface according to claim 5 or 6, characterized in that: In step S2, the coating solution of the ultra-low adsorption cell culture surface coats the surface of the material to be treated to a thickness of 200-500 μm.

8. The method for treating an ultra-low adsorption cell culture surface according to any one of claims 5 to 7, characterized in that: In step S2, the coating solution of the ultra-low adsorption cell culture surface is coated on the surface of the material to be treated by spray coating, immersion coating or spin coating; preferably, the spray coating includes atomization spraying, high-pressure spraying or electrostatic spraying.

9. The method for treating an ultra-low adsorption cell culture surface according to any one of claims 5 to 8, characterized in that: The ultraviolet curing includes using an ultraviolet lamp to irradiate the surface of the material to be treated coated in step S2, the wavelength of the ultraviolet lamp is 254nm or 365nm, and the irradiation time is 10min-60min.

10. An ultra-low adsorption cell culture material obtained by the ultra-low adsorption cell culture surface treatment method according to any one of claims 5 to 9.

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