CN113881562B - Preparation and application of high-flux in-situ observable cell 3D culture plate - Google Patents
Preparation and application of high-flux in-situ observable cell 3D culture plate Download PDFInfo
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- 238000011065 in-situ storage Methods 0.000 title claims abstract description 21
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- 238000004113 cell culture Methods 0.000 claims abstract description 98
- 239000002245 particle Substances 0.000 claims abstract description 26
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- 230000003075 superhydrophobic effect Effects 0.000 claims abstract description 15
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 21
- 238000003756 stirring Methods 0.000 claims description 20
- FFUAGWLWBBFQJT-UHFFFAOYSA-N hexamethyldisilazane Chemical compound C[Si](C)(C)N[Si](C)(C)C FFUAGWLWBBFQJT-UHFFFAOYSA-N 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 13
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 12
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 9
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Abstract
The invention belongs to the field of biomedical materials, and particularly relates to preparation and application of a high-flux in-situ observable cell 3D culture plate. Comprising the following steps: step 1: preparation of alkylated SiO 2 Sol; step 2: washing the cell culture plate; step 3: preparing the alkylated SiO in the step 1 2 And (3) coating the sol on the surface of the cell culture plate in the step (2) to obtain the coated cell culture plate. The application is to prepare liquid marbles with monolayer nano-particle structures on the surface of a coated cell culture plate. The super-hydrophobic SiO prepared by the invention 2 The coating has weak binding force characteristics, and the outermost particles are easy to transfer. By rolling the cell culture fluid droplets directly on the coating, in situ preparation of the liquid marbles within the multi-well plate can be achieved. The prepared liquid marble can be directly used for cell 3D culture and high-throughput detection. Compared with the powder source liquid marble, the liquid marble is more transparent, is more beneficial to observing the internal situation, and avoids the waste and pollution caused by the operations of powder removal, marble transfer and the like.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to preparation and application of a high-flux in-situ observable cell 3D culture plate.
Background
Three-dimensional (3D) cell culture plays an important role in basic research and drug experiments. A great deal of researches show that the three-dimensional cell culture can provide living conditions more similar to living conditions in organisms for cell growth, influence the functions of cell migration, aggregation, proliferation, differentiation and the like, and ensure that cells keep good biochemical and physiological reactions.
In contrast to conventional two-dimensional cell culture, cells divide in all directions in a three-dimensional environment, and their responses to endogenous and exogenous stimuli, such as temperature, PH, nutrient concentration, oxygen content, etc., remain consistent with their responses in vivo.
Conventional two-dimensional cell culture often loses the properties of primary cells, often is incompatible with in vivo conditions, and cannot reflect cell-to-cell and cell-to-matrix interactions. Animal experiments are completely performed in vivo, but become abnormally complex due to the influence and restriction of various factors in vivo and the mutual influence of in vivo and external environments, it is difficult to study a single process, and it is difficult to study an intermediate process.
The three-dimensional cell culture technology well solves the problems, can also enable the micro-environmental parameters of the cells to be easy to control and monitor, and has great value in the aspects of tumor treatment mechanism analysis, drug evaluation, stem cell differentiation, regenerative medicine and the like.
Currently, there are several methods for three-dimensional cell culture, including the use of low adhesion hydrophilic surfaces, rotating-agitation-based bioreactors, magnetic manipulation, and hanging drop techniques, among others. The methods have the related problems of long culture period, damage to cell morphology due to shear stress, incapability of in-situ observation due to inverted cell spheres, high cost and the like.
In recent years, it has been found that using droplets of surface-attached particles, i.e., liquid marbles, as incubators can well avoid the phenomenon of cell adhesion to substrates, thereby achieving 3D growth of cells.
However, the preparation of conventional liquid marbles still has the following problems:
1. the traditional liquid marbles are prepared by taking powder as a particle source, removing redundant powder after the liquid marbles are prepared, and transferring the liquid marbles to a culture dish and other platforms for subsequent operation, so that great limitation is brought to high-throughput cell culture and comparative analysis;
2. the surface particles of conventional powder liquid marbles form clusters of more than tens of microns, resulting in poor transparency of the liquid marbles, often requiring the extraction of internal material for observation.
Disclosure of Invention
Aiming at the technical problems, the invention provides a preparation method of a high-flux in-situ observable cell 3D culture platform, which is based on a common cell culture plate and combines a weak-binding force super-hydrophobic coating and a single-layer nanoparticle structure liquid marble technology, wherein the single-layer nanoparticle structure liquid marble formed in the cell culture plate can effectively block adhesion of cells to a substrate for a long time, so that the cells are suspended above the bottom of the spherical liquid marble, and 3D growth is realized.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a preparation of a high-throughput, in situ observable cell 3D culture plate comprising the steps of:
step 1: preparation of alkylated SiO 2 Sol;
step 2: washing the cell culture plate;
step 3: preparing the alkylated SiO in the step 1 2 And (3) coating the sol on the surface of the cell culture plate in the step (2) to obtain the coated cell culture plate.
Preferably, the specific steps of the step 1 include:
step 1.1: adding tetraethoxysilane into absolute ethanol solution, and continuously stirring at room temperature for 5-20min to completely and uniformly mix the tetraethoxysilane and the absolute ethanol solution to obtain solution A;
step 1.2: adding ammonia water into the solution A obtained in the step 1.1, stirring while adding, continuously stirring at room temperature for 10-40min, standing and aging for 7-10 days to obtain solution B;
step 1.3: adding hexamethyldisilazane into the solution B obtained in the step 1.2, stirring while adding, continuously stirring for 10-40min, standing and aging at room temperature for 1-2 days to obtain alkylated SiO 2 And (3) sol.
Preferably, the porous cell culture plate is soaked in an ethanol solution with the volume fraction of 90% for 30min, taken out, rinsed with ultrapure water for 1-2 times, and then rapidly dried by an air gun for later use.
Preferably, the specific step of the step 3 includes:
step 3.1: the alkylated SiO prepared in step 1.3 is reacted with 2 Adding the sol into each hole of the cell culture plate cleaned in the step 2, slightly shaking to enable the sol to cover the inner walls of the holes of the cell culture plate completely, and then pouring out the sol;
step 3.2: repeating the operation of step 3.1 for 2-3 times, and then rapidly drying with ear washing ball or air gun;
step 3.3: repeating the operation of step 3.2 for 3-6 times to obtain the super-hydrophobic SiO coated on the surface 2 Xerogel coated porous cell culture plates.
Preferably, in the step 1, the volume ratio of the added reagent is: ethyl orthosilicate: absolute ethyl alcohol: ammonia water: hexamethyldisilazane= (4-6): 50: (1.5-2.1): (1.12-4.28).
Preferably, the porous cell culture plate with 12 holes or 24 holes is selected in the step 2, and the porous cell culture plate is made of polystyrene.
Preferably, in the step 3.2, the interval is 10-30s each time the extra sol is poured and new sol needs to be added again.
Preferably, in the step 3.2, when the air gun is used for drying, the air pressure is controlled to be less than 5 standard atmospheric pressures, and the circulation is performed for at least 6 times; when the ear washing ball is used for drying, the cell culture plate with low adhesion can be obtained after 3 times of circulation.
Preferably, the concentration of the ammonia water in the step 1 is 25% -28%.
The use of a high-throughput, in situ observable cell 3D culture plate for the preparation of liquid marbles, comprising the steps of:
step 1.1: adding cell culture solution into coated porous cell culture plate with injector or liquid-transferring gun to form quasi-spherical liquid drop, and gently shaking the culture plate to make the liquid drop roll back and forth in the coated hole, transferring the outermost layer particles of the coating layer to the surface of the liquid drop under the drive of minimized energy to obtain alkylated SiO on the surface 2 Particle-coated monolayer nanostructured liquid marbles;
step 1.2: repeating the operation of step 1.1 until the liquid drops fill each hole of the cell culture plate to obtain the cell culture liquid marbles with the single-layer nano-particle structure, the number of which is the same as that of the holes. Compared with the prior art, the invention has the beneficial effects that:
(1) The invention adopts the evaporation-induced self-assembly film forming technology, is constructed by using the weak binding force super-hydrophobic coating and the liquid marbles with single-layer nano particle structure technology based on the common cell culture plate, and coats a layer of xerogel super-hydrophobic SiO on the bottom and the inner wall of each hole of the cell culture plate 2 The coating has weak binding force, and the outermost particles are easy to transfer to the surface of the liquid drop to form a single-layer nanoLiquid marbles with a rice grain structure. The liquid marble with the monolayer nanoparticle structure can effectively prevent cells from adhering to a substrate for a long time, provides a good platform for 3D culture of the cells, has ultrahigh transparency, and can observe the cells in situ. Therefore, by directly rolling the cell culture solution drops on the coating, the in-situ preparation of the liquid marbles in the porous plate can be realized, and the prepared liquid marbles can be directly used for 3D cell culture and high-throughput detection.
(2) At present, when 3D culturing cells using powder liquid marbles, problems such as removal of excessive powder and transfer of liquid marbles are involved, and furthermore, the powder liquid marbles have poor transparency, so that it is difficult to observe the internal situation. In contrast, the invention is based on the common cell culture plate, combines the weak-binding force super-hydrophobic coating and the single-layer nano-particle structure liquid marble technology, not only can realize the 3D culture of cells, but also provides convenience for the in-situ observation of cells due to the ultrahigh transparency of the single-layer nano-particle structure liquid marble. More importantly, the porous cell culture plate can be used for easily carrying out synchronous culture and behavior comparison of cells in a plurality of liquid marbles under the same environment, which is very beneficial to cell mass production, drug screening, high-throughput cell detection and analysis and the like.
(3) The preparation method provided by the invention has the advantages of simple process and low cost, is suitable for 12-hole, 24-hole and even more-hole cell culture plates, and avoids the waste of materials, labor and time costs, the influence of factors such as cell pollution and the like caused by operations such as powder removal, marble transfer and the like.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
In the drawings:
FIG. 1 is a flow chart of the method of the present invention;
FIG. 2 shows the plated (A) and uncoated (B) areas of the cell culture plate;
FIG. 3 shows the pure water droplet morphology of the plated (A) and unplated (B) areas of the cell culture plate;
FIG. 4 shows the droplet morphology of the cell culture fluid in the plated (A) and uncoated (B) areas of the cell culture plate;
FIG. 5 is a schematic diagram of the preparation of liquid marbles using a coated cell culture plate and for three-dimensional cell culture;
FIG. 6 shows pure water marbles and cell culture medium marbles wrinkled on the surface after liquid extraction;
FIG. 7 shows the MCF7 breast cancer cells cultured for 3 days, wherein the culture vessel is an uncoated cell culture plate (a), a superhydrophobic surface bare droplet (b) and a liquid marble (c) according to the invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention is carried out by alkylating SiO 2 Dripping sol into clean cell culture plate holes, slightly shaking to cover the inner walls of the holes of the cell culture plate, pouring the sol out, rapidly drying wet gel layer in the cell culture plate by using an air gun or ear washing ball in an air blowing mode, and repeating the above process for multiple times to obtain the super-hydrophobic SiO coated with weak binding force 2 Coated cell culture plates. The purpose of repeated steps is to increase the thickness of the coating, improve the lyophobic capacity, and avoid the adhesion of liquid drops so as to ensure the successful preparation of liquid marbles.
Obtained by the methodA physical diagram of the cell culture plate is shown in FIG. 2 (A), and FIG. 2 (B) shows the uncoated area of the cell culture plate. From the figure, the SiO of the bottom and the side wall of the film coating area can be known 2 The super-hydrophobic coating presents rich colors under a film interference mechanism, and is characterized in that the coating is prepared by multiple layers of alkylated SiO 2 The outer-most particles are easily transferred to the surface of the liquid drop, so that the liquid marbles with ultra-high transparency and single-layer nano particle structure are formed, as shown in fig. 3 (A) and 4 (A), and fig. 3 (B) and 4 (B) are respectively the pure water and the liquid drop form of the cell culture liquid in the non-film-plating area of the cell culture plate.
The liquid types in FIG. 3 (A, B) are pure water, and the liquid types in FIG. 4 (A, B) are pink cell culture solutions.
Research shows that the liquid marbles with single-layer nano-particle structures formed in the cell culture plate can effectively block adhesion of cells to a substrate for a long time, so that the cells are suspended above the bottom of the spherical liquid marbles, and 3D growth is realized. In addition, the comparison of cell behavior in a plurality of liquid marbles is easily performed using cell culture plates in combination with conventional detection means, which is very advantageous for high throughput detection assays. The specific technical scheme is as follows:
a preparation of a high-throughput, in situ observable cell 3D culture plate comprising the steps of:
step 1: preparation of alkylated SiO 2 Sol:
step 1.1: adding tetraethoxysilane into absolute ethanol solution, and continuously stirring at room temperature for 5-20min to completely and uniformly mix the tetraethoxysilane and the absolute ethanol solution to obtain solution A;
step 1.2: adding ammonia water into the solution A obtained in the step 1.1, stirring while adding, continuously stirring at room temperature for 10-40min, standing and aging for 7-10 days to obtain solution B;
step 1.3: adding hexamethyldisilazane into the solution B obtained in the step 1.2, stirring while adding, continuously stirring for 10-40min, standing and aging at room temperature for 1-2 days to obtain alkylated SiO 2 And (3) sol.
Step 2: the common polystyrene porous cell culture plate with 12 holes or 24 holes is selected, the porous cell culture plate is soaked in ethanol solution with the volume fraction of 90 percent for 30min, and the ethanol solution with the volume fraction of 70-90 percent has better killing effect on fungi, and the surface of the porous cell culture plate can reach the clean requirement by killing the fungi spores at about 30 percent. Taking out, washing with ultrapure water for 1-2 times, and then rapidly drying by an air gun for later use:
step 3: FIG. 5 is a schematic diagram showing the preparation of liquid marbles using coated cell culture plates and use in 3D cell culture; the method comprises the following specific steps:
step 3.1: the alkylated SiO prepared in step 1.3 is reacted with 2 Adding the sol into each hole of the cell culture plate cleaned in the step 2, slightly shaking to enable the sol to cover the inner walls of the holes of the cell culture plate completely, and then pouring out the sol;
step 3.2: repeating the operation of step 3.1 for 2-3 times, when adding new sol again after pouring excessive sol, keeping the hole in semi-moist state for 10-30s, to obtain the foundation of strong lyophobic ability and weak binding force film, then rapidly drying with ear-washing ball or air gun, and controlling air pressure to be less than 5 standard atmospheric pressures and circulating at least 6 times when drying with air gun; when the ear washing ball is used for drying, the cell culture plate with low adhesion can be obtained by circulating for 3 times;
step 3.3: repeating the operation of step 3.2 for 3-6 times to obtain the super-hydrophobic SiO coated on the surface 2 Xerogel coated porous cell culture plates.
The step 3.2 and the step 3.3 are carried out for multiple times, so as to increase the thickness of the coating, improve the lyophobic capacity, and avoid the adhesion of liquid drops so as to ensure the successful preparation of liquid marbles.
The cell 3D culture plate prepared as described above was applied to the preparation of liquid marbles, comprising the steps of:
step 1.1: adding a certain volume of cell culture solution into the porous cell culture plate by using a syringe or a pipette, adding a part of liquid, gently shaking to form liquid marbles, adding the rest volume of liquid into the liquid marbles, and continuously shaking to obtain a target volume of liquid marbles, so that the adhesion of liquid drops to a substrate can be better avoided, and the liquid is ensuredSuccessfully preparing marbles to form spheroid liquid drops, then gently shaking the culture plate to enable the liquid drops to roll back and forth in the holes of the coating film, transferring particles at the outermost layer of the coating to the surfaces of the liquid drops under the drive of minimized energy, and obtaining SiO with alkylated surfaces 2 Particle-encapsulated monolayer nanostructured liquid marbles.
The liquid marbles with the monolayer nanoparticle structures have ultrahigh transparency, and the particles on the surface of the marbles cannot be directly observed by naked eyes, but the existence of the particles can be verified through the influence of the particles on the surface properties. For this purpose, a part of the liquid may be extracted from the marble, and the marble surface may be significantly wrinkled due to the increase of the particle density, as shown in fig. 6.
The surface of the liquid drop only grabs the outermost particles of the super-hydrophobic coating to form a liquid marble with a single-layer nano particle structure, which is determined by the combination of the film binding force and the surface energy of the liquid drop, and after the surface of the liquid drop is wrapped by the single-layer nano particles, the surface can not be covered with more particles.
The type of liquid in this step may be pure water, aqueous solutions (including salt solutions in water as a solvent, alcohol solutions, cell culture solutions, etc.), glycerin solutions, etc.
Step 1.2: repeating the operation of step 1.1 until the liquid drops fill each hole of the cell culture plate to obtain the cell culture liquid marbles with the single-layer nano-particle structure, the number of which is the same as that of the holes.
Example 1:
a preparation of a high-throughput, in situ observable cell 3D culture plate comprising the steps of:
step 1: preparation of alkylated SiO 2 Sol:
step 1.1: weighing 16mL of tetraethoxysilane, adding the tetraethoxysilane into 200mL of absolute ethanol solution, and continuously stirring at room temperature for 20min to completely and uniformly mix the tetraethoxysilane and the absolute ethanol solution to obtain solution A;
step 1.2: adding 8.4mL of ammonia water with the concentration of 25% -28% into the solution A obtained in the step 1.1, stirring while adding, continuously stirring at room temperature for 40min, standing and aging for 7 days to obtain a solution B;
step 1.3: step toAdding 4.48mL of hexamethyldisilazane into the solution B obtained in the step 1.2, stirring while adding, continuously stirring for 30min, standing at room temperature, aging for 2 days to obtain alkylated SiO 2 And (3) sol.
The invention adopts the proportion of the tetraethoxysilane, the absolute ethyl alcohol, the ammonia water and the hexamethyldisilazane, and can lead SiO to the following 2 Sol with particle size distribution of about 20nm ensures the ultra-high transparency of liquid marbles with single-layer nano-particle structures prepared in later stage, and the xerogel SiO formed by coating 2 The coating has the characteristic of weak binding force, and the outermost particles are easily transferred to the surface of the liquid drops to form liquid marbles.
Step 2: the selected porous cell culture plate of 24-hole polystyrene is soaked in absolute ethyl alcohol for 30min, taken out and washed with ultrapure water for 2 times, and then is quickly dried by an air gun for later use:
step 3: the method comprises the following specific steps:
step 3.1: the alkylated SiO prepared in step 1.3 is reacted with 2 Adding the sol into each hole of the cell culture plate cleaned in the step 2, slightly shaking to enable the sol to cover the inner walls of the holes of the cell culture plate completely, and then pouring out the sol;
step 3.2: repeating the operation of step 3.1 for 2 times, and when adding new sol again after pouring excessive sol, keeping the hole in semi-moist state for 10s, and rapidly drying with air gun, wherein the air pressure is less than 5 standard atmospheric pressures to prevent the sol from being blown away.
Step 3.3: repeating the operation of the step 3.2 for 6 times to obtain the super-hydrophobic SiO with weak binding force coated on the surface 2 Xerogel coated 24 well cell culture plates.
The prepared cell 3D culture plate is applied to the preparation of liquid marbles, and pure water liquid marbles with larger surface tension and larger volume are prepared, and the method comprises the following steps:
step 1.1: measuring 300 mu L of pure water by a liquid-transferring gun, adding the pure water into the porous cell culture plate obtained in the step 3.3 to form spheroid-like liquid drops, then gently shaking the culture plate to enable the liquid drops to roll back and forth in the holes of the coating film, and finally coating the outermost particlesTransfer to the surface of the droplet under the drive of energy minimization to obtain SiO with alkylated surface 2 Particle-coated monolayer nanostructured liquid marbles;
step 1.2: repeating the operation of step 1.1 until the liquid drops fill up each hole of the cell culture plate, and obtaining 24 pure water liquid marbles with the quasi-spherical monolayer nano-particle structure.
The present example demonstrates that a liquid with a large surface tension, such as pure water, can obtain liquid marbles by this simple direct addition method even though the volume is relatively large (300 microliters).
Example 2:
a preparation of a high-throughput, in situ observable cell 3D culture plate comprising the steps of:
step 1: preparation of alkylated SiO 2 The sol was prepared in the same manner as in example 1:
step 2: washing the cell culture plate, the washing method was the same as in example 1;
step 3: the method comprises the following specific steps:
step 3.1: the alkylated SiO prepared in step 1.3 is reacted with 2 Adding the sol into each hole of the cell culture plate cleaned in the step 2, slightly shaking to enable the sol to cover the inner walls of the holes of the cell culture plate completely, and then pouring out the sol;
step 3.2: repeating the operation of step 3.1 for 3 times, and when the new sol needs to be added again each time the extra sol is poured out, keeping the holes in a semi-moist state for 10 seconds, and then blowing with the ear washing ball to quickly dry.
Step 3.3: repeating the operation of the step 3.2 for 3 times to obtain the super-hydrophobic SiO with weak binding force coated on the surface 2 Xerogel coated 24 well cell culture plates.
The prepared cell 3D culture plate is applied to the preparation of liquid marbles, and the preparation of the cell culture liquid marbles with smaller surface tension and smaller volume comprises the following steps:
step 1.1: 200. Mu.L of MCF7 breast cancer cell culture medium (DMEM+10% FBS+1% double antibody) was measured by a pipette, and added to the porous cell culture plate obtained in step 3.3 to form spheroid-like droplets, followed byGently shaking the culture plate to make the liquid drop roll back and forth in the coated hole, and transferring the outermost particles of the coating to the surface of the liquid drop under the drive of minimized energy to obtain SiO with alkylated surface 2 Liquid marbles with single-layer nano-particle structures and wrapped by particles;
step 1.2: repeating the operation of step 1.1 until the liquid drops fill up each hole of the cell culture plate, and obtaining 24 cell culture liquid marbles with the quasi-spherical monolayer nanoparticle structures. After 3 days of culture of MCF7 breast cancer cells in these marbles, a typical cytosphere was formed, as shown in fig. 7. The comparative experiments show that cells cultured in uncoated cell culture plates and superhydrophobic surface bare droplets are in a substantially in-plane agglomerated morphology, which is far from the three-dimensional spheroids of the present invention.
Example 3:
a preparation of a high-throughput, in situ observable cell 3D culture plate comprising the steps of:
step 1: preparation of alkylated SiO 2 The sol was prepared in the same manner as in example 1:
step 2: washing the cell culture plate, the washing method was the same as in example 1;
step 3: plating film on the surface of the cell culture plate, the method is the same as that of example 2;
the prepared high-flux in-situ observable cell 3D culture plate is applied to the preparation of liquid marbles, and the preparation of the cell culture liquid marbles with smaller surface tension and larger volume comprises the following steps:
step 1.1: when the volume is large, the low surface tension liquid droplets easily adhere to the substrate and cannot form liquid marbles. For this, in this example, 100. Mu.L of MCF7 breast cancer cell culture medium (DMEM+10% FBS+1% double antibody) was first measured by a pipette, and then added to the 24-well cell culture plate obtained in step 3.3 to form a spheroid-like droplet, and then the plate was gently shaken to roll the droplet back and forth in the well of the plated film, and the outermost particles of the coating were transferred to the surface of the droplet under the drive of energy minimization to obtain a 100. Mu.L surface alkylated SiO volume 2 Liquid marbles with single-layer nano-particle structures and wrapped by particles; 200. Mu.L of the medium was then added to the above-mentioned mixtureIn 100. Mu.L of liquid marbles, droplets were obtained in which only a partial area of the surface was covered with particles, and the droplets were gently swirled again to completely cover the surface with particles, thereby obtaining 300. Mu.L of cell culture liquid marbles.
Step 1.2: repeating the operation of step 1.1 until the liquid drops fill up each hole of the cell culture plate to obtain 24 cell culture liquid marbles with a spheroid-like monolayer nanoparticle structure, which are used for 3D culture of cells.
The surface tension and volume both have an effect on the successful preparation of liquid marbles, and three examples show the preparation of liquid marbles under conditions of high (pure water) low (cell culture broth) surface tension and large (300. Mu.l) and small (200. Mu.l) volume, respectively.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (4)
1. A preparation method of a high-throughput in-situ observable cell 3D culture plate is characterized by comprising the following steps: the method comprises the following steps:
step 1: preparation of alkylated SiO 2 Sol;
step 2: washing the cell culture plate;
step 3: preparing the alkylated SiO in the step 1 2 Coating the sol on the surface of the cell culture plate in the step 2 to obtain a coated cell culture plate;
the specific steps of the step 1 comprise:
step 1.1: adding tetraethoxysilane into absolute ethanol solution, and continuously stirring at room temperature for 5-20min to completely and uniformly mix the tetraethoxysilane and the absolute ethanol solution to obtain solution A;
step 1.2: adding ammonia water into the solution A obtained in the step 1.1, stirring while adding, continuously stirring at room temperature for 10-40min, standing and aging for 7-10 days to obtain solution B;
step 1.3: adding hexamethyldisilazane into the solution B obtained in the step 1.2, stirring while adding, continuously stirring for 10-40min, standing and aging at room temperature for 1-2 days to obtain alkylated SiO 2 Sol;
the specific operation of the step 2 is as follows: soaking the porous cell culture plate in 90% ethanol solution for 30min, taking out, washing with ultrapure water for 1-2 times, and rapidly drying with air gun;
the specific steps of the step 3 include:
step 3.1: the alkylated SiO prepared in step 1.3 is reacted with 2 Adding the sol into each hole of the cell culture plate cleaned in the step 2, slightly shaking to enable the sol to cover the inner walls of the holes of the cell culture plate completely, and then pouring out the sol;
step 3.2: repeating the operation of step 3.1 for 2-3 times, and then rapidly drying with ear washing ball or air gun;
step 3.3: repeating the operation of step 3.2 for 3-6 times to obtain the super-hydrophobic SiO coated on the surface 2 Xerogel coated porous cell culture plates;
in the step 1, the volume ratio of the added reagent is as follows: ethyl orthosilicate: absolute ethyl alcohol: ammonia water: hexamethyldisilazane= (4-6): 50: (1.5-2.1): (1.12-4.28);
the porous cell culture plate with 12 holes or 24 holes is selected in the step 2, and is made of polystyrene;
in the step 3.2, when the air gun is used for drying, the air pressure is controlled to be less than 5 standard atmospheric pressures, and the circulation is carried out for at least 6 times; when the ear washing ball is used for drying, the cell culture plate with low adhesion can be obtained after 3 times of circulation.
2. The method of preparing a high throughput, in situ observable cell 3D culture plate of claim 1, wherein: in the step 3.2, the interval time is 10-30s when new sol needs to be added again for pouring out the redundant sol.
3. The method of preparing a high throughput, in situ observable cell 3D culture plate of claim 1, wherein: the concentration of the ammonia water in the step 1 is 25% -28%.
4. Use of a high throughput, in situ observable cell 3D culture plate prepared according to the method of claim 1, characterized in that: the cell 3D culture plate is applied to the preparation of liquid marbles, and the specific process comprises the following steps:
step 1.1: adding cell culture solution into a cell 3D culture plate by using a syringe or a liquid-transferring gun to form spheroid liquid drops, then gently shaking the culture plate to enable the liquid drops to roll back and forth in the holes of a coating film, transferring particles at the outermost layer of the coating layer to the surfaces of the liquid drops under the drive of energy minimization to obtain SiO with alkylated surfaces 2 Particle-coated monolayer nanostructured liquid marbles;
step 1.2: repeating the operation of step 1.1 until the liquid drops fill each hole of the cell culture plate to obtain the cell culture liquid marbles with the single-layer nano-particle structure, the number of which is the same as that of the holes.
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