WO2024113490A1

WO2024113490A1 - Well plate for researching interaction between different cell spheres and use thereof

Info

Publication number: WO2024113490A1
Application number: PCT/CN2023/074877
Authority: WO
Inventors: 张秀莉; 曲玥阳; 杨瑒; 季珍妮; 罗勇
Original assignee: 苏州大学
Priority date: 2022-11-30
Filing date: 2023-02-08
Publication date: 2024-06-06
Also published as: CN115926985A

Abstract

A well plate for researching interaction between different cell spheres, comprising at least two types of culture wells with different heights. By means of adjusting the liquid level height of a culture medium in the well plate, the different culture wells are in communication by means of the liquid level, thus realizing the mutual communication between cell spheres in the different culture wells. The well plate is a super-hydrophobic low-adhesion well plate or a hydrophilic low-adhesion well plate; when the well plate is the super-hydrophobic low-adhesion well plate, the bottom surfaces of the culture wells are super-hydrophobic surfaces with micro-nano morphology, and the side surfaces of the culture wells are hydrophobic surfaces or super-hydrophobic surfaces; when the well plate is the hydrophilic low-adhesion well plate, the bottom surfaces of the culture wells are modified with a polymer coating with micro-nano morphology, and the contact angle between the polymer coating and an aqueous phase solution is less than 90°.

Description

A well plate for studying the interaction between different cell spheres and its application

Technical Field

The invention relates to the technical field of three-dimensional cell culture, and in particular to a well plate for studying the interaction between different cell spheres and an application thereof.

Background technique

Organ-organ interaction is a very important research topic in biology. In vitro models are an important tool for studying organ-organ interaction. For example, organ chips are a bionic in vitro model that is often used in organ-organ interaction research. Cell spheroids are another bionic in vitro model that can theoretically also be used for organ-organ interaction research, and using cell spheroids to study organ-organ interaction has its own unique advantages, such as better biomimetic properties. However, in practice, there are not many reports on the use of cell spheroids to study organ-organ interactions. There are reports that after synthesizing cell spheroids using traditional low-adhesion well plates, the cell spheroids are taken out and placed on a microfluidic chip platform, for example, to study the interaction between cell spheroids, but the experimental operation is very cumbersome. A convenient way is to generate cell spheroids directly in low-adhesion well plates, and then directly use the well plates to study the interaction between cell spheroids, but there are almost no reports of such cases. The main reasons are:

First, if you use cell spheroids in a well plate to study organ-organ interactions, you need to put one cell spheroid into the well where the other cell spheroid is located. First, the state of the two cell spheroids in the same well is uncontrollable. They may stick together and fuse into a ball, or they may be too far away and the interaction is not obvious. Second, it is generally uncertain how many cell spheroids can be produced in each well of a low-adhesion well plate, so it is also unclear how many cell spheroids each type has, making it impossible to conduct quantitative research on the interaction between cell spheroids.

Secondly, there are many technical problems in using traditional low-adhesion well plates to produce cell spheroids. The principle of traditional low-adhesion well plates is to modify the surface of the wells with a chemical coating. This layer of chemical coating can prevent cells from adhering to the bottom of the wells, thereby allowing cells suspended in the culture medium to spontaneously aggregate and grow to form three-dimensional cell spheroids or organoids. However, the problem with traditional low-adhesion well plates is that the original cells, primary tissues, or the final aggregated cell spheroids and organoids will continuously secrete extracellular matrix and proteins into the culture medium, and the chemical coating is not resistant to these extracellular matrix and proteins. These extracellular matrix and proteins will settle and adhere to the chemical coating, making the chemical coating ineffective, so that the cells, primary tissue blocks, or the generated cell spheroids and organoids will adhere to the bottom of the wells, thus affecting the three-dimensional culture. The technical problems of traditional low-adhesion well plates lead to three consequences. First, it is not suitable for the three-dimensional culture of all cells, especially those cells that secrete a lot of extracellular matrix and protein (such as kidney cells and nerve cells); second, for most common cells, traditional low-adhesion well plates are generally difficult to achieve long-term three-dimensional culture; third, traditional low-adhesion well plates are generally difficult to use multiple times.

Summary of the invention

The technical problem to be solved by the present invention is to provide a well plate for studying the interaction between different cell spheres. By placing the holes in the well plate at different heights and gradually adding culture medium to submerge the holes at the same height, the time point and duration of the interaction between the cell spheres can be accurately controlled, and the interaction between multiple cell spheres can be studied.

The present invention provides a well plate for studying the interaction between different cell spheres, wherein the well plate comprises at least two culture wells of different heights, and the culture wells are used to culture different types of cell spheres; by adjusting the liquid level of the culture medium in the well plate, different culture wells are connected through the liquid level, thereby realizing the mutual communication between the cell spheres in different culture wells; wherein the well plate is a super hydrophobic low adhesion well plate or a hydrophilic low adhesion well plate;

When the well plate is a super-hydrophobic low-adhesion well plate, the bottom surface of the culture well is a super-hydrophobic surface with a micro-nano morphology, and its contact angle with the aqueous solution is greater than 120°; the side surface of the culture well is a hydrophobic surface or a super-hydrophobic surface with a micro-nano morphology, and its contact angle with the aqueous solution is greater than 90°;

When the well plate is a hydrophilic low-adhesion well plate, the bottom surface of the culture well is modified with a polymer coating with a micro-nano morphology, and the contact angle between the polymer coating and the aqueous solution is less than 90°.

In order to make it possible to study organ-organ interactions using cell spheroids in a well plate, the present invention first controls the number of cells in each well so that they just aggregate into a single cell spheroid; then the culture wells in the well plate are placed at different heights, so that by gradually adding culture fluid to submerge the wells at the same height, the interaction between cell spheroids can be precisely controlled, and the interaction between multiple cell spheroids can be studied.

The present invention provides the following two solutions to the problems that traditional low-adhesion well plates are not suitable for three-dimensional culture of cells with active extracellular matrix and protein secretion, are difficult to achieve long-term three-dimensional culture, and cannot be reused multiple times.

The first method is to provide a super-hydrophobic low-adhesion orifice plate, using a micro-nano super-hydrophobic surface with a self-cleaning function to manufacture a low-adhesion orifice plate. After the aqueous solution (such as culture fluid) is injected into the culture well containing the super-hydrophobic micro-nano structure, the solution cannot completely cover the super-hydrophobic bottom surface, and can only partially contact or even not contact, so as to exist in a semi-suspended or even suspended state, thereby reducing the contact area between the cell and the bottom surface of the culture well. In addition, since this micro-nano super-hydrophobic surface does not adhere to the extracellular matrix and protein, these substances deposited thereon are easily washed off, thereby realizing the super-hydrophobic low-adhesion orifice plate. Repeated and long-term use of the board.

The second method is to provide a new type of hydrophilic low-adhesion well plate, the bottom surface of which has a micro-nanomorphic polymer coating that is hydrophilic, and the contact angle between it and the aqueous solution (such as culture medium) is less than 90°. This micro-nanostructured polymer coating can prevent cells in micro-tissues from crawling out. The principle is: first, the surface of the polymer coating itself is resistant to both cell adsorption and protein adsorption, so these substances deposited on it can be easily washed off, thereby enabling repeated and long-term use of the low-adhesion well plate; second, the polymer coating has a surface micro-nanostructure, similar to hills, and the protein settles down and falls into the gullies, and will not be directly exposed on the surface, thus further increasing the bottom surface's ability to prevent cell adhesion and crawling out.

Furthermore, the material for making the orifice plate can be a polymer material, such as polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polystyrene (PS), polycarbonate (PC), etc.; it can also be a material such as metal, ceramic, glass, quartz, silicon, etc.; or a combination of one or more of the above materials.

In the present invention, the number of culture wells on the orifice plate is not limited, for example, it can be 1-2000. There is no limitation on the shape and flatness of the bottom surface of the culture well, for example, it can be circular, square, rectangular, triangular or other irregular shapes, or it can be an uneven shape. Similarly, there is no limitation on the size and depth of the culture well, which can be set as needed. For example, the depth of the culture well can be 0.5-20 cm, and the diameter can be 0.5-20 cm.

The super hydrophobic low adhesion orifice plate has a manufacturing method comprising method 1 and method 2:

The method 1 comprises the following steps:

S1. Preparation of super hydrophobic substrate;

S2. Preparing a hydrophobic plate with through holes;

S3. The super-hydrophobic bottom plate and the hydrophobic plate are bonded and sealed together to obtain the super-hydrophobic low-adhesion orifice plate;

The second method comprises the following steps:

S4. Obtaining culture wells on the plate by one-step molding;

S5. The inner surface of the culture hole is super-hydrophobic modified to obtain the super-hydrophobic low-adhesion well plate;

Among them, the methods for forming a super-hydrophobic surface include surface etching, MEMS processing, surface enrichment of silica micro-nanoparticles, surface spraying of super-hydrophobic coatings and secondary molding.

In one embodiment, the method for surface enrichment of silica micro-nanoparticles is:

Nano-silicon dioxide particles, n-hexane and chloroform are mixed and then ultrasonically treated to disperse the nano-silicon dioxide particles in the mixed solution; then, a PMMA plate is immersed in the mixed solution, taken out and dried, thereby obtaining a super-hydrophobic surface with a micro-nano structure.

Preferably, the method for enriching the silicon dioxide micro-nano particles on the surface is as follows: weigh 0.25-2g of hydrophobic nano-silicon dioxide particles with a diameter of 2-200nm, measure 20-500ml of n-hexane and 0.5-50ml of chloroform. Mix the above materials and perform ultrasonic treatment to completely disperse the nano-silicon dioxide particles in the mixed solution. Immerse the PMMA plate in the above solution for about 5-300 seconds, take it out and dry it, and a PMMA surface with a surface super-hydrophobic micro-nano structure is obtained.

In another embodiment, the surface etching method is:

The PDMS sheet is immersed in tetraethyl orthosilicate to make it swell, and then the swollen PDMS sheet is taken out and placed in an ethylenediamine aqueous solution; then the PDMS sheet is taken out, rinsed and heat-treated to obtain a super-hydrophobic surface with a micro-nano structure.

Preferably, the surface etching method is as follows: immersing the PDMS sheet in tetraethyl orthosilicate at 30-70° C. for 10-200 minutes, then taking out the swollen PDMS sheet from the tetraethyl orthosilicate solution and immediately floating it in a 5%-40% ethylenediamine aqueous solution. After 3-24 hours, taking it out and rinsing it with deionized water for 3-5 times, and heat-treating it in an oven for 0.5-5 hours, to obtain a PDMS sheet with a surface super-hydrophobic micro-nano structure.

Furthermore, the method of the secondary mold processing is:

a. pouring a prepolymer of an elastic resin (eg, a PDMS prepolymer) onto a template having a surface super-hydrophobic micro-nanostructure, polymerizing the elastic resin prepolymer into a first elastic solid, and then peeling the first elastic solid from the template;

b. silanization modification of the surface of the first elastic solid, and using the silanization modification of the first elastic solid as a template, and then pouring a prepolymer of an elastic resin to polymerize the elastic resin prepolymer into a second elastic solid;

c. Peeling the second elastic solid from the silanized first elastic solid template, at which point a surface super-hydrophobic micro-nano structure is formed on the surface of the second elastic solid.

Furthermore, the template with the surface super-hydrophobic micro-nano structure is a natural material (such as cicada wings, lotus leaves, etc.) or is prepared by artificial methods, and the artificial methods include surface etching, MEMS processing, surface enrichment of silica micro-nano particles, and surface spraying of super-hydrophobic coatings.

For the hydrophilic low-adhesion orifice plate, the method for preparing the polymer coating comprises the following steps:

S1. Mixing glycidyl methacrylate and other polymerizable monomers in water, adding tetramethylethylenediamine and an initiator, and performing a copolymerization reaction; after the reaction, the copolymer solution is dialyzed to obtain a polymer coating liquid;

S2. Pre-treating the well plate to generate polar groups on the bottom surface of the culture wells, and then placing the pre-treated well plate in the polymer coating liquid for immersion treatment; then taking out the well plate, washing off the polymer coating liquid on the surface, and after drying, forming a polymer coating on the bottom surface of the well plate;

Among them, the other polymerizable monomers include one or more of 2-(2-methoxyethoxy)ethyl methacrylate, methoxyethyl methacrylate, methyl dimethacrylate, hexafluorobutyl acrylate, and methyl methacrylate; the polar groups include one or more of hydroxyl, amino, and carboxyl groups.

Furthermore, in step S1, the mixing time of glycidyl methacrylate and other polymerizable monomers in water is 5-30 minutes to ensure uniform mixing.

Furthermore, in step S1, the concentration of tetramethylethylenediamine added is 0.1%-3%, and the concentration of the initiator added is 0.1%-3%.

Furthermore, in step S1, the initiator may be a commonly used initiator in the art, preferably potassium persulfate. The copolymerization reaction time is preferably 30 minutes to 1 day.

Furthermore, in step S1, the dialysis is specifically: using a dialysis membrane to completely dialyze the copolymer solution for two days, changing the solution every 6-12 hours to remove monomers and small molecules that have not undergone polymerization reaction, and the solution in the dialysis bag is the polymer coating solution.

In step S2 of the present invention, by introducing groups such as hydroxyl, amino, carboxyl, etc. on the bottom surface of the culture well, the hydrophilic modification liquid can be bonded to the bottom surface of the culture well by covalent bonds, so that it is firmly bonded to the bottom surface of the culture well to form a polymer coating with micro-nano morphology. Preferably, the well plate is treated with plasma to introduce polar groups on the surface of the well plate. For example, oxygen plasma treatment is used to introduce hydroxyl groups.

Furthermore, in step S2, the soaking temperature is 30-70°C, and the soaking time is 30 minutes to 5 days; the drying temperature is 0-75°C, and the drying time is 30 minutes to 5 days.

The super-hydrophobic low-adhesion well plate provided by the present invention has a bottom surface of a culture well that resists both cell adsorption and protein adsorption. When a solution containing cells is injected into the culture well, the solution cannot completely cover the bottom surface of the culture well, but can only partially contact or even not contact the bottom surface of the culture well. Thus, it exists in a semi-suspended or even suspended state, so the cells are in a suspended culture state, and the cells can be suspended in the culture medium and grow spontaneously in clusters. Therefore, it can be used for three-dimensional culture of cells such as cell spheres, primary tissue blocks, and organoids, as well as for the culture of suspended cells such as blood cells and immune cells. In addition, it can also be used for drug development evaluation, including efficacy, pharmacokinetics, and toxicity.

The hydrophilic low-adhesion well plate provided by the present invention has a bottom surface of a culture well that resists both cell adsorption and protein adsorption. When a solution containing cells is injected into the culture well, the cells are in a state of suspension culture, and the cells can be suspended in the culture fluid and grow spontaneously in clusters. Therefore, it can be used for three-dimensional culture of cells such as cell spheres, primary tissue blocks, and organoids, and can also be used for the culture of suspended cells such as blood cells and immune cells. In addition, it can also be used for drug development evaluation, including efficacy, pharmacokinetics, and toxicity.

The invention also provides application of the well plate in studying the interaction between different cell spheres.

Compared with the prior art, the present invention has the following beneficial effects:

1. The low-adhesion well plate of the present invention comprises at least two wells of different heights, and different types of cell spheres (organoids) can be cultured in the culture wells. Therefore, by adjusting the volume of the culture medium in the well plate, that is, by gradually adding culture medium to submerge the wells at the same height, the cell spheres in the culture wells can communicate with each other, thereby accurately controlling the interaction between the cell spheres.

2. After the aqueous solution is injected into the culture wells, the super-hydrophobic low-adhesion well plate of the present invention cannot completely cover the bottom surface of the culture wells, but can only partially contact or even not contact, so that it exists in a semi-suspended or even suspended state, thereby reducing the contact area between the cells and the bottom surface of the wells. In addition, the surface of the culture wells does not adhere to the extracellular matrix and protein, so these substances deposited thereon can be easily washed off, thereby realizing repeated and long-term use of the well plate.

3. The hydrophilic low-adhesion well plate provided by the present invention has a surface of the culture wells on which extracellular matrix and protein do not adhere, so these substances deposited thereon are easily washed off and can be used for a long time and multiple times, thereby reducing the cost of three-dimensional cell culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the adhesion and disintegration of primary renal tissue blocks on the surface of a traditional low-adhesion plate;

FIG2 is a diagram showing the adhesion and disintegration of primary brain tissue blocks on the surface of a traditional low-adhesion well plate;

FIG3 is an electron microscope photograph of the surface of polydimethylsiloxane after it has been molded twice through a lotus leaf;

FIG4 shows the state of a droplet on a super-hydrophobic PDMS surface;

FIG5 shows the steps of making a polydimethylsiloxane silicon sheet with through holes;

FIG6 is a schematic diagram of the principle of a super hydrophobic low adhesion orifice plate based on polydimethylsiloxane;

FIG7 shows the state of the aqueous solution in the super-hydrophobic pores of PDMS;

FIG8 is a bright field photograph of tumor spheres in super-hydrophobic pores of PDMS;

FIG9 shows the change in contact angle of the superhydrophobic pore after repeated use for 12 times;

FIG10 is a surface electron microscope photograph of a polydimethylsiloxane sheet of silicon dioxide microbubbles;

FIG11 is an electron microscope photograph of a superhydrophobic PMMA surface;

FIG12 is a diagram showing the state of the aqueous solution in the PMMA-PDMS super-hydrophobic pores;

FIG13 is a bright field photograph of tumor spheres in PMMA-PDMS super-hydrophobic pores;

FIG14 shows the state of a droplet in a PMMA super-hydrophobic hole;

FIG15 is a bright field photograph of tumor spheres in PMMA superhydrophobic pores;

FIG16 is a schematic diagram of the structure of a two-layer four-cell spheroid culture well plate;

FIG17 is a photo of a two-layer four-spheroid culture well plate in Example 4;

FIG18 is a fluorescent photograph of a single hepatocyte sphere;

FIG19 is a fluorescent photograph of a single cardiac cell sphere;

FIG20 is the result of the detection of doxorubicin cardiac cell cytotoxicity;

FIG21 is an atomic force microscope image of the polymer coating surface;

FIG22 shows the adsorption of fluorescent proteins on modified PDMS surface (A) and unmodified PDMS surface (B);

FIG23 shows tumor spheres formed in a polydimethylsiloxane polymer coated well plate;

FIG24 is a schematic diagram of a two-layer four-spheroid culture well plate;

FIG25 is a photo of a two-layer four-spheroid culture well plate in Example 6;

Figure 26 is a fluorescent photograph of a single hepatocyte sphere;

FIG27 is a fluorescent photograph of a single cardiac cell sphere;

FIG. 28 shows the results of the doxorubicin cardiac cell cytotoxicity test.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and/or" used herein includes any and all combinations of one or more related listed items.

Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

Comparative Example 1: Adhesion and disintegration of primary kidney and brain tissue blocks on the bottom surface of a traditional low-adhesion plate

The primary tissue blocks of mouse kidney and brain were cultured in Corning's commercial low-adhesion well plates. In the initial state, due to the low adhesion on the bottom of the well plate, the primary tissue blocks of kidney and brain were in a suspended culture state, and their morphology could be well maintained. However, on the fifth day of culture, the primary tissue blocks of kidney and brain adhered to the bottom of the well plate, and the cells crawled out and the tissue blocks disintegrated, as shown in Figures 1 and 2.

Example 1: Using natural materials as templates to make super-hydrophobic low-adhesion orifice plates

1. The preparation method of super hydrophobic low adhesion orifice plate is as follows:

Lotus leaf is fixed on glass plate, liquid polydimethylsiloxane is poured on its surface, spend the night, make liquid polydimethylsiloxane polymerize into solid, the polydimethylsiloxane sheet of solidification is peeled off from lotus leaf as the template of next step.The polydimethylsiloxane template surface is silanized again, then liquid polydimethylsiloxane is poured on template, heat up and polymerize, the newly solidified polydimethylsiloxane sheet is peeled off from the polydimethylsiloxane sheet template of silanization modification.The electron microscope picture on the polydimethylsiloxane sheet surface is as shown in Figure 3, and it has super-hydrophobic nanostructure, contact angle>120 ° (Fig. 4), and the super-hydrophobic polydimethylsiloxane base plate is finished.

The template is prepared by 3D printing technology to make a polydimethylsiloxane plate with holes. The specific process is shown in Figure 5.

The above-mentioned super-hydrophobic polydimethylsiloxane bottom plate and the polydimethylsiloxane plate with holes are sealed using a plasma cleaner to obtain the final cell three-dimensional culture well plate, in which the bottom surface of the hole is polydimethylsiloxane with a surface super-hydrophobic micro-nano structure, and the side surface is hydrophobic polydimethylsiloxane. The schematic diagram of its structure is shown in Figure 6.

When water is injected into the hole, as shown in FIG7 , the water presents an obvious droplet shape.

2. Testing of super hydrophobic low adhesion well plate

The square holes on the super hydrophobic low adhesion plate have a side length of 4 mm and a depth of 4 mm. The plate was used for a tumor cell spheroidization experiment, and human embryonic lung fibroblasts (MRC-5) and human lung adenocarcinoma cells (NCI-H1792) were mixed and cultured in RPMI1640 + 10% FBS. The culture ratio of fibroblasts to cancer cells was 1:1, and the culture environment was 37°C, 5% CO ₂ . After 3 days of culture, tumor cells successfully spheroidized in the super hydrophobic low adhesion plate (Figure 8), achieving an effect similar to that of a traditional low adhesion plate.

The test repeatability showed that the plate was added with various cell culture media for 48 h, then poured out, cleaned, and dried. The experiment was repeated 12 times, and the contact angle of the aqueous solution was measured again, which was still greater than 120° (Figure 9), indicating that the plate can be reused at least 12 times, with a continuous use time of more than 24 days.

Example 2: Making a super-hydrophobic low-adhesion orifice plate using polydimethylsiloxane and polymethyl methacrylate materials

A piece of polydimethylsiloxane plate was immersed in tetraethyl orthosilicate at 50°C for 20 minutes. Then, the polydimethylsiloxane plate swollen with tetraethyl orthosilicate was taken out from the tetraethyl orthosilicate solution and immediately floated in an aqueous solution of 10% ethylenediamine, and silica microbubbles gradually formed on the surface of the polydimethylsiloxane plate. After 10 hours, the polydimethylsiloxane plate with silica microbubbles was taken out and rinsed with deionized water 3 times. Finally, the polydimethylsiloxane plate with silica microbubbles was heat treated in an oven for 1 hour. The electron microscope photo of the prepared superhydrophobic surface is shown in Figure 10.

A method for making a polymethyl methacrylate plate with holes by laser engraving, wherein the holes are circular, and the inner wall of the holes is superhydrophobic modified as follows:

(1) Weigh 0.25 g of hydrophobic nano-silica particles with a diameter of 20 nm, measure 50 ml of n-hexane and 2.5 ml of chloroform, mix them and then perform ultrasonic treatment to make the nano-silica particles completely dispersed in the mixed solution; (2) Immerse a polymethyl methacrylate plate with through holes in the above solution for about 15 seconds, take it out and dry it, and the surface electron microscope photograph of the obtained super-hydrophobic PMMA is shown in Figure 11.

The superhydrophobic polydimethylsiloxane plate and the superhydrophobic polymethyl methacrylate plate with through holes are pressed together to obtain the final cell three-dimensional culture well plate. The bottom of the well is polydimethylsiloxane with a surface superhydrophobic micro-nano structure, and the side is superhydrophobic polymethyl methacrylate. The morphology of the aqueous solution in the well is shown in Figure 12, which is in an obvious droplet state.

2. Testing of super hydrophobic low adhesion well plate

The diameter of the circular hole of the super hydrophobic low adhesion plate is 3 mm and the depth is 4 cm. The plate was used for the tumor cell spheroidization experiment, and the experimental conditions were consistent with those in Example 1. After 3 days of culture, tumor spheroidization was successful in the super hydrophobic low adhesion plate (Figure 13), achieving an effect similar to that of the traditional low adhesion plate.

The test repeatability showed that the well plate was added with cell culture medium (1640 culture medium + 10% fetal bovine serum) for 48 hours, then poured out, washed, dried, and the above experiment was repeated 12 times. Finally, the contact angle of the aqueous solution was measured to be 135.8°, which was still greater than 120°, indicating that the well plate can be reused at least 12 times, and the continuous use time is more than 24 days.

Example 3: Making a super-hydrophobic low-adhesion orifice plate using polymethyl methacrylate material

A polymethyl methacrylate plate with holes was made by laser engraving, wherein the holes were square and the inner wall of the holes was modified to be super-hydrophobic as follows: (1) 0.25 g of hydrophobic nano-silica particles with a diameter of 20 nm were weighed, 50 ml of n-hexane and 2.5 ml of chloroform were measured, and the mixture was subjected to ultrasonic treatment to completely disperse the nano-silica particles in the mixed solution; (2) the polymethyl methacrylate plate with through holes was immersed in the above solution for about 15 seconds, and then taken out and dried. Another polymethyl methacrylate plate was modified by the same method.

The superhydrophobic polymethyl methacrylate plate and the superhydrophobic polymethyl methacrylate plate with through holes are pressed together with bolts to obtain the final cell three-dimensional culture well plate. The bottom of the well is a polymethyl methacrylate plate with a surface superhydrophobic micro-nano structure, and the side is also a superhydrophobic polymethyl methacrylate. The morphology of the aqueous solution in the well is shown in Figure 14.

2. Testing of super hydrophobic low adhesion well plate

The square hole of the super hydrophobic low adhesion plate has a side length of 4 mm and a depth of 4 cm. The plate was used for a tumor cell spheroidization experiment under the same experimental conditions as in Example 2. After 3 days of culture, tumor spheroidization was successful in the super hydrophobic low adhesion plate (Figure 15), achieving an effect similar to that of a conventional low adhesion plate.

The test repeatability showed that the well plate was added with cell culture medium (1640 culture medium + 10% fetal bovine serum) for 48 hours, then poured out, washed, dried, and the above experiment was repeated 12 times. Finally, the contact angle of the aqueous solution was measured to be 132.6°, which was still greater than 120°, indicating that the well plate can be reused at least 12 times, and the continuous use time exceeds 24 days.

Example 4: Two-layer four-well spheroid culture plate

The processing material of the orifice plate is PMMA, the structure is shown in Figure 16, and the actual photo is shown in Figure 17. There are 4 holes, the two holes in the middle are low, and the two holes on the sides are high. The shape of the hole is square, the side length is 2mm, the height of the lowest hole is 800μm, the second highest is 3600μm, and the height of the entire orifice plate is 5mm.

About 1000 liver cells were added to the two middle wells to culture hepatospheres (the hepatocytes and stellate cells induced by IPS were mixed in a ratio of 3:1, and the culture medium was basal culture medium + enzymatic casein + hydrocortisone + L-glutamic acid and other added factors), and about 1000 heart cells were added to the two wells on both sides to culture cardiac spheres (IPS were induced to form cardiomyocytes into spheres, and the culture medium was basal culture medium + enzymatic casein + hydrocortisone + L-glutamic acid + epidermal growth factor, and the culture environment was 37°C, 5% CO ₂ ), the obtained single cardiac cell spheres and single liver cell spheres are shown in Figures 18 and 19 .

Then add empty culture medium to the well plate, covering all four wells. At this time, the cardiac cell spheres and liver cell spheres are in the same culture system and can interact with each other. At this time, add doxorubicin to measure the cardiotoxicity of doxorubicin, and use the doxorubicin toxicity of cardiac cell spheres alone as a control. It can be seen that when cardiac cell spheres and liver cell spheres are cultured at the same time, the cardiotoxicity of doxorubicin is greater, thus confirming the interaction between cardiac cell spheres and liver cell spheres (Figure 20).

Example 5: Polydimethylsiloxane polymer coated orifice plate

Methyl methacrylate and glycidyl methacrylate were uniformly mixed in water for 10 minutes, 0.1% tetramethylethylenediamine and 0.1% potassium persulfate were added to initiate copolymerization for 30 minutes, and the copolymer solution was completely dialyzed for two days using a dialysis membrane, with the solution changed every 12 hours to remove monomers and small molecules that did not undergo polymerization reaction. The solution in the dialysis bag was the polymer coating solution.

A hole plate was made using polydimethylsiloxane material. First, hydroxyl groups were generated on the surface of the hole plate through oxygen plasma. Then, the polymer coating liquid was immersed in the hole and treated at 30°C for 1 hour. Then, the polymer coating liquid was removed, washed three times with deionized water, and dried at 75°C for 1 hour.

First, the bottom of the well plate was characterized by atomic force microscopy. It can be clearly seen from Figure 21 that after modification, there is a layer of polymer on the surface of the well bottom, and the polymer forms a nano-groove structure on the surface.

Then, the anti-protein adsorption characterization was carried out. The fluorescently labeled secondary antibody solution was injected into the modified and unmodified well plates respectively. It can be clearly seen that the fluorescence intensity was very high in the unmodified well plate, indicating that the protein adsorption was very serious, while in the modified well plate, almost no fluorescence was detected, indicating that the coating effectively prevented the adsorption of proteins (Figure 22).

Finally, anti-cell adhesion characterization was performed. Human embryonic lung fibroblasts (MRC-5) and human lung adenocarcinoma cells (NCI-H1792) were mixed and cultured in RPMI 1640 + 10% FBS. The culture ratio of fibroblasts to cancer cells was 1:1, and the culture environment was 37°C and 5% CO _2. After 3 days, the cells did not adhere to the wall, but formed tumor spheres (Figure 23), confirming the ability of the coating to prevent cell adhesion. In the same well, 5 consecutive tumor sphere experiments were performed, all of which were successful, proving the durability of the low adhesion well.

Example 6: Two-layer four-well spheroid culture plate

The processing material of the orifice plate is polydimethylsiloxane, the structure is shown in Figure 24, and the actual picture is shown in Figure 25. There are 4 holes, the two middle holes are low, and the two holes on both sides are high. The shape of the holes is square with a side length of 2mm. The height of the lowest hole is 800μm, and the second highest is 3600μm. The height of the entire orifice plate is 5mm.

Add about 1000 hepatocytes to each of the two middle wells to culture hepatocyte spheres (IPS-induced hepatocytes and Stellate cells were mixed in a ratio of 3:1, the culture medium was basal culture medium + enzymatic casein + hydrocortisone + L-glutamate and other added factors), about 1000 heart cells were added to the two wells on both sides, and heart cell spheres were cultured (IPS were induced to form cardiomyocytes into spheres, the culture medium was basal culture medium + enzymatic casein + hydrocortisone + L-glutamate + epidermal growth factor, the culture environment was 37°C, 5% _CO2 ), and the obtained single heart cell spheres and single liver cell spheres are shown in Figures 26 and 27.

Then, empty culture medium was added to the well plate to cover all four wells, so that the cardiac cell spheres and the hepatocyte spheres could interact with each other in the same culture system. At this time, doxorubicin was added to measure the cardiotoxicity of doxorubicin, and the doxorubicin toxicity of the cardiac cell spheres alone was used as a control. It can be seen that when the cardiac cell spheres and the hepatocyte spheres were cultured at the same time, the cardiotoxicity of doxorubicin was greater, thus confirming the interaction between the cardiac cell spheres and the hepatocyte spheres (Figure 28).

The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

A well plate for studying the interaction between different cell spheres, characterized in that the well plate comprises at least two culture wells of different heights, and the culture wells are used to culture different types of cell spheres; by adjusting the liquid level of the culture medium in the well plate, different culture wells are connected through the liquid level, thereby realizing mutual communication between the cell spheres in different culture wells; wherein the well plate is a super hydrophobic low adhesion well plate or a hydrophilic low adhesion well plate;

When the well plate is a super-hydrophobic low-adhesion well plate, the bottom surface of the culture well is a super-hydrophobic surface with a micro-nano morphology, and its contact angle with the aqueous solution is greater than 120°; the side surface of the culture well is a hydrophobic surface or a super-hydrophobic surface, and its contact angle with the aqueous solution is greater than 90°;

When the well plate is a hydrophilic low-adhesion well plate, the bottom surface of the culture well is modified with a polymer coating with a micro-nano morphology, and the contact angle between the polymer coating and the aqueous solution is less than 90°.
A well plate for studying the interaction between different cell spheres according to claim 1, characterized in that the material used to make the well plate includes one or more of polymers, metals, ceramics, glass, and silicon, and the polymer includes one or more of polymethyl methacrylate, polydimethylsiloxane, polystyrene, and polycarbonate.
The well plate for studying the interaction between different cell spheres according to claim 1, characterized in that the method for making the super hydrophobic low adhesion well plate comprises method one and method two:

The method 1 comprises the following steps:

S1. Preparation of super hydrophobic substrate;

S2. Preparing a hydrophobic plate with through holes;

S3. The super-hydrophobic bottom plate and the hydrophobic plate are bonded and sealed together to obtain the super-hydrophobic low-adhesion orifice plate;

The second method comprises the following steps:

S4. forming culture wells on the plate;

S5. The inner surface of the culture hole is super-hydrophobic modified to obtain the super-hydrophobic low-adhesion well plate;

Among them, the method of forming a super-hydrophobic surface includes at least one of surface etching, MEMS processing, surface enrichment of silicon dioxide micro-nano particles, surface spraying of super-hydrophobic coating and secondary molding.
The well plate for studying the interaction between different cell spheres according to claim 3, characterized in that the method for enriching the silica micro-nanoparticles on the surface is:

Nano-silica particles, n-hexane and chloroform were mixed and then ultrasonically treated to disperse the nano-silica particles in the Then, the PMMA plate is immersed in the mixed solution, taken out and dried, and a super-hydrophobic surface with a micro-nano structure is obtained.
The well plate for studying the interaction between different cell spheres according to claim 3, characterized in that the surface etching method is:

The PDMS sheet is immersed in tetraethyl orthosilicate to make it swell, and then the swollen PDMS sheet is taken out and placed in an ethylenediamine aqueous solution; then the PDMS sheet is taken out, rinsed and heat-treated to obtain a super-hydrophobic surface with a micro-nano structure.
The well plate for studying the interaction between different cell spheres according to claim 3, characterized in that the secondary mold remodeling method is:

a. pouring a prepolymer of an elastic resin onto a template having a surface super-hydrophobic micro-nano structure, polymerizing the prepolymer of the elastic resin into a first elastic solid, and then peeling the first elastic solid from the template;

b. silanization modification of the surface of the first elastic solid, and using the silanization modification of the first elastic solid as a template, and then pouring a prepolymer of an elastic resin to polymerize the elastic resin prepolymer into a second elastic solid;

c. Peeling the second elastic solid from the silanized first elastic solid template, at which point a surface super-hydrophobic micro-nano structure is formed on the surface of the second elastic solid.
A well plate for studying the interaction between different cell spheres according to claim 6, characterized in that the template having a surface super-hydrophobic micro-nano structure is a natural material or is prepared by an artificial method, and the artificial method includes surface etching, MEMS processing, surface enrichment of silica micro-nano particles, and surface spraying of super-hydrophobic coating.
The well plate for studying different cell-spheroid interactions according to claim 1, characterized in that in the hydrophilic low-adhesion well plate, the method for preparing the polymer coating comprises the following steps:

S1. Mixing glycidyl methacrylate and other polymerizable monomers in water, adding tetramethylethylenediamine and an initiator, and performing a copolymerization reaction; after the reaction, the copolymer solution is dialyzed to remove unpolymerized monomers and initiator to obtain a polymer coating liquid;

S2. Pre-treating the well plate to generate polar groups on the bottom surface of the culture wells, and then placing the pre-treated well plate in the polymer coating liquid for immersion treatment; then taking out the well plate, washing off the polymer coating liquid on the surface, and after drying, forming a polymer coating on the bottom surface of the well plate;

Wherein, the other polymerizable monomers include one or more of 2-(2-methoxyethoxy)ethyl methacrylate, methoxyethyl methacrylate, methyl dimethacrylate, hexafluorobutyl acrylate, and methyl methacrylate; the polar group Includes one or more of hydroxyl, amino, and carboxyl groups.
The well plate for studying different cell-spheroid interactions according to claim 8, characterized in that in step S1, the added concentration of tetramethylethylenediamine is 0.1%-3%, and the added concentration of the initiator is 0.1%-3%;

In step S2, the soaking temperature is 30-70°C, and the soaking time is 30 minutes to 5 days; the drying temperature is 0-75°C, and the drying time is 30 minutes to 5 days.
Use of the well plate according to any one of claims 1 to 9 in studying the interactions between different cell spheres.