A method for plasma-modified cell culture plate

Abstract

The present invention relates to a method for plasma-modified cell culture plates, comprising the following steps: S1: placing the cell culture plate in a plasma surface treatment device, evacuating the device, and then introducing a first fluorine-containing compound gas, wherein the first fluorine-containing compound gas is ionized to produce positive and negative charges, and the cell culture plate is subjected to a preliminary surface hydrophobic modification treatment. S2: ending the first fluorine-containing compound gas, introducing a second fluorine-containing compound gas into the plasma surface treatment device, wherein the second fluorine-containing compound gas is ionized to produce positive and negative charges, and the cell culture plate is subjected to a deep surface hydrophobic modification treatment. The modification method of the present invention has short processing time, simple operation, high processing efficiency, low cost, can be used and produced on a large scale, can greatly reduce the surface energy of the cell culture plate, achieves low cell adhesion, and further reduces cell adhesion. In addition, after the fluorine-containing group of the fluorine-containing compound is introduced into the surface of the cell culture plate, it will not harm the growth of the cells, and has high safety.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for plasma-modified cell culture plates. Background Art

[0002] The method of improving the properties of the surface of a substance by physical or chemical methods is called surface modification. The main methods of commonly used surface modification include physical modification and chemical modification. Physical modification includes surface coating modification, surface vacuum plating, sputtering, surface mechanical modification and spraying, etc. Chemical modification includes discharge, radiation, ion plating, electroplating, flame modification, solution treatment, etc.

[0003] Adherent cells have high requirements for the hydrophilicity of the surface of the culture plate wells, so the cell culture plate generally needs to be hydrophilic. Suspended cells require that the cells adhere to the wall as little as possible, and the surface of the cell culture plate is required to have a low surface energy so that the cells cannot attach, that is, an ultra-low adsorption cell culture plate is required. Although the cell culture plate without any treatment can keep most cells in a suspended state, more than about 2% of the cells still adhere to the wall. This less adherence phenomenon can still have an adverse effect on some specific applications. For example, during the stem cell culture process, if very few cells adhere to the wall, it will still cause attachment-mediated differentiation.

[0004] In the prior art, the cell culture plate is generally treated with an anti-cell adhesion liquid. Specifically, the anti-cell adhesion liquid is prepared, then added to the cell culture plate, and evaporated and dried, so that a layer of anti-cell adhesion substance is coated on the surface of the cell culture plate hole, so that the cells cannot adhere. However, this method has a complicated process and a long processing time, which limits large-scale production and has low production efficiency. In addition, the cost of the anti-cell adhesion liquid is high, which further limits its large-scale application.

[0005] Therefore, there is a need to provide a method for modifying and treating cell culture dishes that is lower in cost, simpler to handle, and can be applied on a large scale. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In order to solve the problems in the prior art that the treatment of cell culture plates with anti-cell adhesion liquid is complicated, the treatment efficiency is low, and large-scale application is impossible, the present invention provides a method for plasma-modified cell culture plates.

[0008] (II) Technical solution

[0009] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0010] A method for plasma-modified cell culture plates, comprising the following steps:

[0011] S1: placing an untreated cell culture plate in a plasma surface treatment device, evacuating the plate, and then introducing a first fluorine-containing compound gas, wherein the first fluorine-containing compound gas is ionized to produce positive and negative charges in the plasma surface treatment device, and a preliminary surface hydrophobic modification treatment is performed on the cell culture plate;

[0012] S2: After step S1 is completed, the first fluorine-containing compound gas is stopped from being introduced, and the second fluorine-containing compound gas is introduced into the plasma surface treatment equipment. The second fluorine-containing compound gas is ionized into positive and negative charges in the plasma surface treatment equipment to perform deep surface hydrophobic modification treatment on the cell culture plate.

[0013] The method for plasma-modified cell culture plates as described above preferably further comprises the following steps:

[0014] S3: Repeat steps S1-S2 once to continue the surface hydrophobic modification treatment of the cell culture plate.

[0015] In the method for plasma modification of a cell culture plate as described above, preferably, the first fluorine-containing compound gas and the second fluorine-containing compound gas are one of carbon tetrafluoride, difluoromethane, trifluoromethane or chlorotrifluoromethane.

[0016] In the method for plasma modification of a cell culture plate as described above, preferably, the first fluorine-containing compound gas and the second fluorine-containing compound gas are different fluorine-containing compounds.

[0017] In the method for plasma-modified cell culture plates as described above, preferably, the cell culture plates are made of polystyrene, polypropylene, polyester, polycarbonate, polyethylene or high-density polyethylene.

[0018] In the method for plasma modification of cell culture plates as described above, preferably, in step S1, after evacuation and before introduction of the first fluorine-containing compound gas, the vacuum degree in the plasma surface treatment equipment is 0.02-0.03 mbar.

[0019] In the method for plasma-modified cell culture plates as described above, preferably, in step S1, the initial surface hydrophobic modification treatment of the cell culture plates lasts for 3-7 minutes;

[0020] In step S2, the deep surface hydrophobic modification treatment of the cell culture plate lasts for 8-12 minutes.

[0021] In the method for plasma-modified cell culture plates as described above, preferably, in step S1 and step S2, after the first fluorine-containing compound gas or the second fluorine-containing compound gas is filled, the vacuum degree is 10-20 mbar, the processing temperature is 20-40°C, the processing power is 600-1800 W, and the gas flow rate is 100-300 sccm.

[0022] (III) Beneficial effects

[0023] The beneficial effects of the present invention are:

[0024] First, the growth of cells requires the presence of water. The researchers of the present invention have found that when the hydrophobicity of the culture plate surface is further improved, the contact between water and the cell culture plate will be less, and the cell adhesion phenomenon will be further reduced. Therefore, the present invention reduces the occurrence of cell adhesion by improving the hydrophobicity of the cell culture plate.

[0025] Secondly, the present invention improves the surface of the cell culture plate through the fluorine-containing groups of the fluorine-containing compound gas to achieve the improvement of the hydrophobic performance of the cell culture plate. Specifically, the present invention passes the fluorine-containing compound gas into the plasma surface treatment equipment, and ionizes the fluorine-containing compound to generate fluorine-containing groups in the plasma state. The surface energy of the fluorine-containing groups is extremely low, and complex chemical reactions occur on the surface of the cell culture plate, introducing the fluorine-containing groups into the surface of the cell culture plate. The fluorine-containing groups perform surface hydrophobic modification on the cell culture plate on the surface of the cell culture plate, so that the hydrophobic performance of the surface of the cell culture plate is greatly improved.

[0026] Thirdly, the present invention uses the first fluorine-containing compound gas and the second fluorine-containing compound gas respectively to perform surface hydrophobic modification on the cell culture plate through a two-step modification method. Compared with the single-step modification treatment, the present invention uses a two-step modification method to reduce the steric hindrance effect between the groups, so that the fluorine-containing groups can modify the surface of the cell culture plate more fully, the hydrophobic performance is better, and the wall adhesion phenomenon is less.

[0027] The plasma modified cell culture plate method of the present invention has a short treatment time, simple operation, high treatment efficiency, low cost, can be used and produced on a large scale, can greatly reduce the surface energy of the cell culture plate, achieves low cell adhesion, and further reduces cell adhesion. In addition, the fluorine-containing group of the fluorine-containing compound is not toxic after being introduced into the surface of the cell culture plate, will not harm the growth of cells, and has high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the contact angle of a water drop on a cell culture plate after plasma modification in Example 1 of the present invention;

[0029] Figure 2Schematic diagram of the contact angle of a water drop on a cell culture plate without any treatment in Comparative Example 2;

[0030] Figure 3 It is a comparison chart of the number of adherent cells in the cell culture plates treated with hydrophobic modification, hydrophilic treatment and untreated surfaces in Example 1.

[0031] [Description of Reference Numerals]

[0032] 1: water drop; 2: cell culture plate. DETAILED DESCRIPTION

[0033] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0034] Cell growth requires the presence of water. The researchers of the present invention have found that when the hydrophobicity of the culture plate surface is further improved, the contact between water and the cell culture plate will be less, and the cell adhesion phenomenon will be further reduced. Therefore, the present invention reduces the occurrence of cell adhesion by improving the hydrophobicity of the cell culture plate. Specifically, the present invention provides a method for plasma-modified cell culture plates, comprising the following steps:

[0035] S1: Place the untreated cell culture plate in a plasma surface treatment device and evacuate the area to prevent the gas in the air from ionizing other groups in the plasma state and affecting the cell culture plate. After reaching the specified vacuum degree, introduce the first fluorine-containing compound gas, which ionizes positive and negative charges in the plasma surface treatment device to perform preliminary surface hydrophobic modification on the cell culture plate.

[0036] S2: After step S1 is completed, the first fluorine-containing compound gas is stopped from being introduced, and the second fluorine-containing compound gas is introduced into the plasma surface treatment equipment. The second fluorine-containing compound gas is ionized into positive and negative charges in the plasma surface treatment equipment to perform deep surface hydrophobic modification treatment on the cell culture plate.

[0037] The fluorine-containing groups generated by the ionization of the fluorine-containing compound gas improve the surface of the cell culture plate, which can improve the hydrophobic performance of the cell culture plate. Specifically, the present invention introduces the fluorine-containing compound gas into a plasma surface treatment device, so that the fluorine-containing compound in the plasma state generates fluorine-containing groups, the surface energy of which is extremely low, and complex chemical reactions can occur on the surface of the cell culture plate, and the fluorine-containing groups are introduced into the surface of the cell culture plate, which has the effect of hydrophobic modification treatment on the surface of the cell culture plate, so that the hydrophobic performance of the surface of the cell culture plate is greatly improved.

[0038] In addition, the present invention divides the modification process of the cell culture plate by the fluorine-containing gas into two steps, and the surface hydrophobic modification of the cell culture plate is performed by the first fluorine-containing compound gas and the second fluorine-containing compound gas respectively. Compared with the single-step modification process, the two-step modification method reduces the steric hindrance effect between the groups, so that the modification of the surface of the cell culture plate by the fluorine-containing group is more sufficient, the hydrophobic performance is better, and the wall adhesion phenomenon is less.

[0039] Preferably, in order to further enhance the degree of surface hydrophobic modification treatment and improve the hydrophobic properties of the cell culture plate, steps S1-S2 may be repeated once after step S2 to continue to perform surface hydrophobic modification treatment on the cell culture plate.

[0040] The first fluorine-containing compound gas and the second fluorine-containing compound gas are one of carbon tetrafluoride, difluoromethane, trifluoromethane or trifluorochloromethane. Preferably, the first fluorine-containing compound gas and the second fluorine-containing compound gas are different fluorine-containing compounds.

[0041] The cell culture plate can be made of polystyrene, polypropylene, polyester, polycarbonate, polyethylene or high-density polyethylene. In terms of structure and shape, the cell culture plate can be a 6-, 12-, 24-, 48- or 96-well plate, the well bottom of the cell culture plate can be a flat bottom, a round bottom, a U-shaped bottom or a V-shaped bottom, and the cell culture plate can be covered or uncovered.

[0042] Preferably, in step S1, after evacuation and before the first fluorine-containing compound gas is introduced, the vacuum degree in the plasma surface treatment equipment is maintained at 0.02-0.03 mbar.

[0043] The time of surface hydrophobic modification treatment should be controlled within a reasonable range. Specifically, in step S1, the initial surface hydrophobic modification treatment time of the cell culture plate is 3-7min, preferably 5min. In step S2, the depth surface hydrophobic modification treatment time of the cell culture plate is 8-12min, preferably 10min. If the treatment time is too short, less than the minimum value of the above-mentioned treatment time, it will cause uneven surface treatment of the cell culture plate, insufficient hydrophobic performance, and many cells will still adhere to the wall. If the treatment time is too long, exceeding the maximum value of the above-mentioned prescribed time, it will cause excessive surface treatment of the cell culture plate, which will not only cause a waste of energy, but also cause structural damage to the cell culture plate, affecting subsequent use.

[0044] In the specific modification process of step S1 and step S2, after the first fluorine-containing compound gas or the second fluorine-containing compound gas is filled, the vacuum degree is maintained at 10-20 mbar, the processing temperature is 20-40°C, the processing power is 600-1800 W, and the gas flow rate is 100-300 sccm.

[0045] Compared with the prior art, the plasma modified cell culture plate method of the present invention has a short treatment time, simple operation, high treatment efficiency, low cost, can be used and produced on a large scale, can greatly reduce the surface energy of the cell culture plate, achieves low cell adhesion, and further reduces cell adhesion. In addition, the fluorine-containing group of the fluorine-containing compound is not toxic after being introduced into the surface of the cell culture plate, will not harm the growth of cells, and has high safety.

[0046] Example 1

[0047] This embodiment provides a method for plasma-modified cell culture plates, comprising the following steps:

[0048] S1: Place the untreated uncovered 96-well cell culture plate in the plasma surface treatment equipment, close the hatch, turn on the vacuum pump, and start evacuation. After the vacuum degree in the treatment chamber of the plasma surface treatment equipment reaches 0.03 mbar, open the difluoromethane inlet valve, treat for 5 minutes, and then close the difluoromethane inlet valve.

[0049] S2: After closing the difluoromethane inlet valve, open the tetrafluorocarbon inlet valve and the treatment time is 10 minutes.

[0050] In the above step S1 and step S2, after filling with difluoromethane or carbon tetrafluoride, the vacuum degree is maintained at 20 mbar, the processing temperature is 25° C., the processing power is 1000 W, and the gas flow rate is 200 sccm.

[0051] Example 2

[0052] This embodiment provides a method for plasma-modified cell culture plates, comprising the following steps:

[0053] S1: Place the untreated uncovered 96-well cell culture plate in the plasma surface treatment equipment, close the hatch, turn on the vacuum pump, and start evacuation. After the vacuum degree in the treatment chamber of the plasma surface treatment equipment reaches 0.03 mbar, open the trifluoromethane inlet valve, treat for 5 minutes, and then close the trifluoromethane inlet valve.

[0054] S2: After closing the trifluoromethane inlet valve, open the tetrafluorocarbon inlet valve and the treatment time is 10 minutes.

[0055] In the above step S1 and step S2, after filling with trifluoromethane or carbon tetrafluoride, the vacuum degree is maintained at 20 mbar, the processing temperature is 25° C., the processing power is 1000 W, and the gas flow rate is 200 sccm.

[0056] Example 3

[0057] This embodiment provides a method for plasma-modified cell culture plates, comprising the following steps:

[0058] S1: Place the untreated uncovered 96-well cell culture plate in the plasma surface treatment equipment, close the hatch, turn on the vacuum pump, and start evacuation. After the vacuum degree in the treatment chamber of the plasma surface treatment equipment reaches 0.03 mbar, open the trifluorochloromethane inlet valve, treat for 5 minutes, and then close the trifluorochloromethane inlet valve.

[0059] S2: After closing the trifluorochloromethane inlet valve, open the tetrafluorocarbon inlet valve and the treatment time is 10 minutes.

[0060] In the above step S1 and step S2, after filling with chlorotrifluoromethane or carbon tetrafluoride, the vacuum degree is maintained at 20 mbar, the processing temperature is 25° C., the processing power is 1000 W, and the gas flow rate is 200 sccm.

[0061] Example 4

[0062] This embodiment provides a method for plasma-modified cell culture plates, comprising the following steps:

[0063] S1: Place the untreated uncovered 96-well cell culture plate in the plasma surface treatment equipment, close the hatch, turn on the vacuum pump, and start evacuation. After the vacuum degree in the treatment chamber of the plasma surface treatment equipment reaches 0.03 mbar, open the difluoromethane inlet valve, treat for 5 minutes, and then close the difluoromethane inlet valve.

[0064] S2: After closing the difluoromethane inlet valve, open the tetrafluorocarbon inlet valve and the treatment time is 10 minutes.

[0065] S3: Open the difluoromethane inlet valve, treat for 5 minutes, and then close the difluoromethane inlet valve.

[0066] S4: After closing the difluoromethane inlet valve, open the tetrafluorocarbon inlet valve and the treatment time is 10 minutes.

[0067] In the above steps S1-S4, after filling with difluoromethane or carbon tetrafluoride, the vacuum degree is maintained at 20 mbar, the processing temperature is 25° C., the processing power is 1000 W, and the gas flow rate is 200 sccm.

[0068] Comparative Example 1

[0069] This comparative example provides a method for plasma-modified cell culture plates, which differs from comparative example 1 in that only one fluorine-containing compound gas, carbon tetrafluoride, is used to perform surface hydrophobic modification treatment on the cell culture plates for 15 minutes.

[0070] Comparative Example 2

[0071] This comparative example provides a 96-well cell culture plate without any treatment.

[0072] Water drops were added to the cell culture plates in Examples 1-3 and Comparative Examples 1-2, and the contact angles between the water drops and the cell culture plates were measured. The contact angles quantitatively represent the hydrophilicity and hydrophobicity of the cell culture plates. When the contact angle is less than 90°, the smaller the contact angle, the better the hydrophilicity; when the contact angle is greater than 90°, the larger the contact angle, the better the hydrophobicity.

[0073] Contact angle tests were performed on Examples 1-3 and the comparative example. The test results are shown in Table 1.

[0074]

[0075] It can be seen from Table 1 that the hydrophobic property of the cell culture plate treated with surface hydrophobic modification is significantly better than that of the untreated cell culture plate in Comparative Example 2. The surface hydrophobic modification effect of the cell culture plate treated with two gases in two steps is better than that of the surface hydrophobic modification treated with a single gas in Comparative Example 1. This may be because the step-by-step treatment of the two gases can reduce the steric hindrance effect between the molecular groups, so that the fluorine-containing groups can treat the polymer surface of the cell culture plate more fully.

[0076] The cell culture plates in Example 1, which were subjected to hydrophilic modification (TC treatment), and in Comparative Example 2, which were not subjected to any treatment, were selected for cell culture verification:

[0077] 1. First, take out a cryopreservation tube containing Vero cells from the liquid nitrogen tank and quickly put it into a 37℃ water bath for two minutes.

[0078] 2. During the two-minute recovery period, add 15 mL of fresh culture medium (10% FBS, 90% DMEM) to the cellpro T75 cell culture flask.

[0079] 3. After two minutes of resuscitation, take out the Vero cell cryopreservation tube from the water bath and wipe it with 75% alcohol for disinfection.

[0080] 4. Take the cells under sterile conditions, use a 1000μL pipette tip to suck the frozen cell solution into a 15mL centrifuge tube, add 5mL of culture medium, centrifuge and remove the supernatant. Then add 1mL of culture medium to resuspend. Add 1mL of cell culture medium to the cellpro T75 cell culture flask, shake gently, and place it in an incubator at 37℃ and 5% CO2 for static culture.

[0081] 5. After culturing for 2 days, digest the cells and collect them. The collected cells are plated at 1x10 4 The cells were inoculated evenly on the above three cell culture plates, with 5 wells inoculated on each cell culture plate.

[0082] 6. After 24 hours, take out the three cell culture plates, discard the supernatant, digest the bottom adherent cells, and then record the number of adherent cells in the ultra-low adsorption 96-well cell culture plate, the TC-treated 96-well cell culture plate, and the 96-well cell culture plate without any treatment, see Table 2.

[0083]

[0084] As shown in Table 2, the adhesion effect of Vero cells in the ultra-low adsorption cell culture plate was reduced by 98.97% compared with the TC-treated culture plate, and the adhesion effect was also reduced by 82.57% compared with the culture plate without any treatment, indicating that the ultra-low adsorption culture plate has a significant effect on inhibiting cell adhesion.

[0085] The above embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention. Those skilled in the art may make various deformations or modifications within the scope of the claims, which belong to the essential content of the present invention.

Claims

1. A method for plasma-modified cell culture plates, characterized in that: The steps include: S1: placing an untreated cell culture plate in a plasma surface treatment device, evacuating the device, and then introducing a first fluorine-containing compound gas, wherein the first fluorine-containing compound gas is ionized to produce positive and negative charges in the plasma surface treatment device, and fluorine-containing groups are introduced into the surface of the cell culture plate, thereby performing a preliminary surface hydrophobic modification treatment on the cell culture plate; S2: After step S1 is completed, the first fluorine-containing compound gas is stopped from being introduced, and the second fluorine-containing compound gas is introduced into the plasma surface treatment device, and the second fluorine-containing compound gas is ionized into positive and negative charges in the plasma surface treatment device, and the fluorine-containing groups are introduced into the surface of the cell culture plate, and the cell culture plate is subjected to deep surface hydrophobic modification treatment; The first fluorine-containing compound gas is difluoromethane or trifluoromethane, and the second fluorine-containing compound gas is carbon tetrafluoride; The cell culture plate is made of polystyrene, polypropylene, polyester, polycarbonate, polyethylene or high-density polyethylene; In step S1, the initial surface hydrophobic modification treatment of the cell culture plate lasts for 3-7 minutes; In step S2, the deep surface hydrophobic modification treatment of the cell culture plate lasts for 8-12 minutes; In step S1 and step S2, after the first fluorine-containing compound gas or the second fluorine-containing compound gas is filled, the vacuum degree is 10-20 mbar, the processing temperature is 20-40° C., the processing power is 600-1800 W, and the gas flow rate is 100-300 sccm.

2. The method for plasma-modified cell culture plates according to claim 1, characterized in that: The following steps are also included: S3: Repeat steps S1-S2 once to continue the surface hydrophobic modification treatment of the cell culture plate.

3. The method for plasma-modified cell culture plates according to claim 1, characterized in that: In step S1, after evacuation and before the first fluorine-containing compound gas is introduced, the vacuum degree in the plasma surface treatment equipment is 0.02-0.03 mbar.

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