WO2025157093A1 - Culture device, method for processing culture device, and cell culture method - Google Patents

Abstract

Disclosed in the present invention are a culture device, a method for processing a culture unit, and a cell culture method. The culture device comprises a culture box and a culture unit. The culture box has an accommodating space and also has an opening at the top. The culture unit is made of polydimethylsiloxane and is located in the accommodating space, the bottom surface of the culture unit closely fits with the bottom wall of the culture box, and the side wall of the culture unit closely fits with the inner wall of the culture box. The culture unit is provided with a culture recess, an input hole and an output hole. The culture recess is recessed from the bottom surface of the culture unit. The input hole is recessed from the top wall of the culture recess to the top surface of the culture unit. The output hole is recessed from the top wall of the culture recess to the top surface of the culture unit and spaced apart from the input hole.

Description

Culture device, culture vessel processing method and cell culture method

Cross-reference to related applications

This patent application claims priority to the Chinese patent application filed on January 22, 2024, with application number 202410090207.6 and invention name “Culture device, processing method of culture vessel and cell culture method”. The full text of the above application is incorporated herein by reference.

Technical Field

The technical field of cell culture devices, in particular, relates to a culture device, a processing method of a culture vessel and a cell culture method.

Background Art

With the rapid development of tissue engineering and regenerative medicine, more and more research requires the isolation, culture, and expansion of primary cells. However, traditional primary tissue isolation and stem cell culture methods have multiple problems, such as low cell viability, unstable tissue fixation methods, and low cloning efficiency. The success rate is even lower if the primary culture is performed on a small amount of tissue. Conventional methods require large amounts of culture medium and reagents, which increases experimental costs and limits the feasibility of large-scale experiments. Therefore, researchers need to seek more efficient and cost-effective culture methods to improve experimental efficiency and reduce resource consumption.

Summary of the Invention

In order to solve the above technical problems, embodiments of the present invention provide a culture device, a processing method of a culture vessel, and a cell culture method to solve the above problems.

In order to solve the above technical problems, the embodiment of the present utility model provides a culture device, which includes:

a culture box having a receiving space and an open top; and

A culture vessel made of polydimethylsiloxane and located in the accommodation space, wherein the bottom surface of the culture vessel is in close contact with the bottom wall of the culture box, and the side wall of the culture vessel is in close contact with the inner side wall of the culture box, and the culture vessel is provided with:

a culture tank, the culture tank being formed by a depression in the bottom surface of the culture vessel;

an input hole, the input hole being formed by the top wall of the culture tank being recessed into the top surface of the culture vessel; and

An output hole is formed by the top wall of the culture tank being recessed to the top surface of the culture vessel and is spaced apart from the input hole.

The present invention also relates to a method for processing a culture vessel, comprising the steps of:

S1. Mixing the silicon polymer and the cross-linking agent in proportion and stirring evenly to form a mixed colloid;

S2, extracting bubbles in the mixed colloid;

S3, stacking the sheet-shaped mold on the bottom wall of the culture box, then pouring the mixed colloid after the bubbles are extracted into the culture box, and heating and drying until the mixed colloid is solidified to form a culture container;

S4. After taking out the culture vessel, the mold is separated from the culture vessel, and a culture tank is formed on the bottom surface of the culture vessel; an input hole and an output hole are punched on the top wall of the culture tank with a puncher, and the input hole and the output hole pass through the culture vessel respectively.

In one embodiment, the culture box is a culture dish, and the mold is a glass slide.

In one embodiment, in step S2, a plurality of the molds are stacked on the bottom wall of the culture box and spaced apart;

In step S4, the plurality of molds are separated from the culture vessel to form a plurality of culture tanks, and the input holes and the output holes are punched out on the top walls of the plurality of culture tanks using a puncher.

In one embodiment, at least two of the molds are stacked vertically.

In one embodiment, at least two of the moulds are of different sizes.

In one embodiment, in step S3, the heating temperature is 70°-80° and the heating time is 60 min-70 min.

The present invention also relates to a cell culture method comprising the steps of:

a. Cleaning the culture device according to claim 2 to remove impurities and residues on the surface of the culture device;

b. sterilizing the incubator and the culture box; c. placing the incubator in the culture box;

d. Move the culture medium from the input port into the culture tank.

The cell culture method according to claim 8, characterized in that the culture vessel is made of polydimethylsiloxane; in step a, the culture vessel is placed in a phosphate buffered saline solution and soaked for a preset time to remove impurities and residues on the surface of the culture vessel, and then the culture vessel is placed in deionized water for ultrasonic cleaning to ensure the cleanliness of the culture dish surface.

In one embodiment, in step b, after the incubator is sterilized, the incubator needs to be wiped dry using sterile filter paper.

In response to these problems, the present invention has developed a culture vessel made of elastic transparent material for fixing tissues and culturing primary cells. The device uses a small-volume culture tank and breathable materials, which can reduce the risk of contamination and reduce the consumption of culture medium. The use of this device can better fix tiny tissues, improve the survival rate and proliferation rate of primary cells, and provide tissues with microenvironmental conditions that are closer to their native state. By optimizing the fixation and culture methods, the present invention is expected to improve the effect of tissue fixation and culture, and provide a more reliable experimental platform for cytological and biological research. In addition, the small culture tank can also reduce the use of culture medium and containers, thereby reducing costs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of an incubator according to an embodiment of the present invention.

FIG2 is a cross-sectional view of the incubator of the embodiment shown in FIG1 .

FIG3 is a schematic diagram of an incubator according to another embodiment of the present invention.

FIG3 a is a schematic diagram of an incubator according to another embodiment of the present invention.

FIG4 a shows the composition of the culture medium for primary culture of mouse brain tissue.

FIG4 b is a graph showing cell growth on the fourth day of primary culture of mouse brain tissue in three sizes of culture vessels and 24-well plates.

FIG5 is a comparison of the cell yields of primary cultures of mouse brain tissue in three sizes of incubators and 24-well plates.

FIG6 a is a graph comparing the number of cells harvested and the total number of cells per unit area in three sizes of culture vessels and 24-well plates.

FIG6 b is a comparison chart of the cell harvest number and the total cell number in three specifications of culture vessels and 24-well plates under unit volume of culture medium.

FIG7 is a diagram showing the effects of neurosphere culture of primary cultured cells of mouse brain tissue on the eighth day in three specifications of culture vessels and 24-well plates.

Figure 8 shows the culture of dental pulp tissue in a 24-well plate and a 0.17 mm deep culture tank.

FIG9 shows the positive clone rate and the number of cells per unit volume and per unit area of dental pulp tissue cultured in a 24-well plate and a 0.17 mm deep culture tank.

Figure 10 shows the culture of dental pulp tissue in a 96-well plate and a 0.2 mm deep culture tank.

Figure 11 shows the culture conditions of dental pulp tissue in a 96-well plate and a 0.2 mm deep culture tank and the cell crawling-out ratio.

Figure 12 shows the cell counts of dental pulp tissue in a 96-well plate and a 0.2 mm deep culture tank.

FIG13 shows the cell crawling-out ratio of dental pulp tissue in a 96-well plate and a 0.2 mm deep culture tank.

FIG14 shows the cell count of dental pulp tissue in a 96-well plate and a 0.2 mm deep culture tank.

Figure 15 shows cells of dental pulp tissue on the sixth day in a 96-well plate and a 0.2 mm deep culture tank.

Figure numerals: 100, culture vessel; 1, culture tank; 2, input hole; 3, output hole.

DETAILED DESCRIPTION

To make the objectives, technical solutions, and advantages of the present invention more apparent, various embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, those skilled in the art will appreciate that many technical details are provided in various embodiments of the present invention to facilitate a better understanding of the present application. However, even without these technical details and the various variations and modifications based on the following embodiments, the technical solutions claimed in the claims of this application can be implemented.

Unless the context requires otherwise, throughout the specification and claims, the word "comprise" and variations such as "include" and "have" should be construed in an open, inclusive sense, that is, should be interpreted to mean "including, but not limited to."

The following will describe in detail various embodiments of the present invention in conjunction with the accompanying drawings to provide a clearer understanding of the objectives, features and advantages of the present invention. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the present invention, but are only intended to illustrate the essential spirit of the technical solution of the present invention.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is generally employed in its sense including "and/or" unless the context clearly dictates otherwise.

In the following description, in order to clearly show the structure and working mode of the present invention, many directional words will be used for description, but words such as "front", "back", "left", "right", "outside", "inside", "outward", "inward", "up", and "down" should be understood as convenient terms and should not be understood as restrictive terms.

The present invention relates to a culture device and a culture vessel 100 thereof. The culture device includes a culture box and a culture vessel 100, wherein the culture box has a storage space and a top opening. The culture box can optionally include but is not limited to a 24-well plate, a 96-well plate, a 12-well plate, a 6-well plate and a culture dish, etc. The present invention does not limit the specific implementation of the culture box.

The culture vessel 100 is made of an elastic transparent material, which includes rubber, preferably polydimethylsiloxane. Polydimethylsiloxane is non-toxic and has the characteristics of ultra-high molecular weight, low viscosity and unique fluidity. The culture vessel 100 made of polydimethylsiloxane also has certain toughness, good compatibility and high transparency, and is very suitable for culturing cells.

The incubator 100 can be placed in a culture dish, or six incubators 100 can be placed in six wells of a 6-well plate. Taking the culture dish as an example, the bottom surface of the incubator 100 is in close contact with the bottom wall of the culture dish, and the side wall of the incubator 100 is in close contact with the inner wall of the culture box.

Specifically, the culture vessel 100 can be a column, such as a cylindrical structure or a quadrangular prism structure. The present invention does not limit the specific structure of the culture vessel 100. The structure of the culture vessel 100 only needs to match the accommodation space of the culture box.

In the embodiment shown in Figures 1 and 2, the incubator 100 is a cylindrical structure that can be placed inside a culture dish. The dimensions of the incubator 100 can be adjusted based on the outer diameter of the culture dish, i.e., the outer diameter of the incubator 100 must match the inner diameter of the culture dish. The height of the incubator 100 is preferably less than the depth of the culture dish, and the height range of the incubator 100 is preferably 0.5 cm to 1 cm, including but not limited to 0.5 mm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, and 1 cm. During the manufacturing process of the incubator 100, the appropriate thickness facilitates the demolding of the incubator 100 from the culture dish and allows the incubator 100 to fit seamlessly into the culture dish, with virtually no culture medium exchange between the individual culture troughs. The incubator 100 comprises a culture trough 1, an input port 2, and an output port 3. The culture trough 1 is formed by a depression in the bottom surface of the incubator 100. The inner wall of the culture trough 1 can be a quadrangular prism or other cylindrical structure. The depth of the culture trough 1 can also be adjusted as desired. The specific shape of the culture trough 1 is not limited by the present invention.

In the embodiment of Fig. 1 and Fig. 2, culture tank 1 is a cylindrical structure. The depth range of this culture tank 1 is preferably 0.1mm-2mm, and optionally includes 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm and 2mm. In addition, in the embodiment shown in Fig. 1 and Fig. 3, the bottom surface of incubator 100 is provided with three culture tanks 1, and three culture tanks 1 are evenly spaced and have the same shape. It should be understood that more culture tanks 1 can also be provided, and the shapes of multiple culture tanks 1 can be different, and the depth of multiple culture tanks 1 can also be provided as required, to adapt to different cell culture needs.

As a preferred embodiment, as shown in Figure 3, the three culture tanks 1 are all elongated quadrangular prism structures, and the length of each culture tank 1 is greater than its height. The length of the culture tank 1 can be set according to the radial dimension of the culture vessel 100, as long as it is not greater than the radial dimension of the culture vessel 100. The width of the culture tank 1 can also be set according to the number of culture tanks 1, and multiple culture tanks 1 can be set at intervals and do not exceed the outer diameter of the culture vessel 100. In the embodiment shown in Figure 3a, among the multiple culture tanks 1, some of the culture tanks 1 are elongated quadrangular prism structures, and there are also cylindrical structures in the embodiment shown in Figure 1. Of course, in other embodiments, the shape of the culture tank 1 can also be set to other shapes, and the present invention does not limit the specific shape of the culture tank 1.

Input hole 2 is a through hole extending vertically. It can be considered a through hole formed by the top wall of culture tank 1 being recessed into the top surface of incubator 100. Input hole 2 is the passage through which culture medium enters culture tank 1. Culture medium can be added to culture tank 1 from input hole 2 via a pipette. The diameter of input hole 2 preferably ranges from 0.5 mm to 1.5 mm. During pipetting, the pipette can expand input hole 2. After pipetting, input hole 2 is squeezed and becomes smaller, facilitating easy transfer of culture medium into culture tank 1 while preventing excessive air or impurities from entering culture tank 1 through input hole 2.

The output hole 3 and the input hole 2 are formed in basically the same way, both of which are formed by being recessed from the top wall of the culture tank 1 to the top surface of the culture vessel 100. The output hole 3 is spaced apart from the input hole 2 and has a larger diameter than the input hole 2. During the cell culture process, the nutrient solution needs to be replaced. When changing the liquid, the nutrient solution can be added to the culture tank 1 from the input hole 2 through a pipette, and the old nutrient solution can be discharged from the output hole 3. Therefore, in order to facilitate the smooth discharge of the liquid from the output hole 3, the diameter of the output hole 3 needs to be larger than the diameter of the input hole 2. As a preferred embodiment, the diameter range of the output hole 3 is 1.5mm-2.5mm. The output hole 3 within this range can discharge the liquid smoothly, and can also avoid excessive evaporation of the culture medium during the cell culture process, and can also prevent impurities from entering the culture tank 1 from the output hole 3.

Each culture tank 1 is connected to an input hole 2 and an output hole 3. Each input hole 2 and output hole 3 are formed by the top wall of the corresponding culture tank 1 being recessed into the top surface of the incubator 100. The input hole 2 and output hole 3 of each culture tank 1 are spaced apart. In other words, the number of input holes 2, output holes 3, and culture tanks 1 corresponds one to one.

The present invention also relates to a method for processing the incubator 100, which specifically comprises the following steps:

S1. Mix the silicone polymer and the cross-linking agent in a certain proportion and stir them evenly to form a mixed colloid. The silicone polymer and the cross-linking agent are generally in a ratio of 10:1.

S2. Vacuum extraction is performed on the mixed colloid to remove bubbles in the mixed colloid to ensure that the mixed colloid is free of bubbles;

S3. Overlay the sheet mold on the bottom wall of the incubator, then inject the colloid mixture after degassing into the incubator until the colloid mixture covers the sheet mold. Then, heat the colloid mixture on a metal heat block at 70°-80° for 60-70 minutes. Heat and dry until the colloid mixture solidifies and forms the incubator 100.

The thickness of the mixed colloid injected into the culture box primarily affects the thickness of the culture container 100, that is, its height. The thickness of the culture container 100 determines its hardness. A thinner culture container 100 is softer, making it easier to remove from the mold, punch holes, and perform other subsequent operations. A thicker culture container 100 is harder and less convenient to remove from the mold, but it allows for better separation of the holes, ensuring their individuality. Therefore, the thickness of the culture container 100 is preferably between 0.5 cm and 1 cm.

S4. After removing the incubator 100, separate the mold from the incubator 100. Separate the solidified incubator 100 from the incubator box and carefully remove the incubator 100, taking care not to damage the incubator box. Then, carefully remove the mold and other components using tweezers. After the mold is removed, a culture tank 1 is formed on the bottom surface of the incubator 100. Use a punch to punch an input hole 2 and an output hole 3 on the top wall of the culture tank 1, ensuring that the input hole 2 and the output hole 3 extend through the incubator 100.

In step S4, the plurality of molds are separated from the culture vessel 100 to form a plurality of culture tanks 1, and the input holes 2 and the output holes 3 are punched out on the top walls of the plurality of culture tanks 1 using a puncher.

In step S3, the culture box can be a multi-well plate or a culture dish. Selecting an appropriate outer mold size can ensure the compatibility of the mold with the cell culture equipment and provide sufficient culture area.

Taking the culture dish as an example, the sheet-shaped mold can be made of glass slides or other metal sheets. Multiple glass slides are placed at intervals on the bottom wall of the culture dish. To increase the thickness of the culture tank 1, two, three, or more glass slides can be stacked vertically. Of course, it is also possible to stack some glass slides, while other single glass slides are stacked on the bottom wall of the culture dish, to form culture tanks 1 of varying depths. Molds of different sizes can also be used to form culture tanks 1 of varying sizes. By adjusting the size and shape of the mold, the mold can be customized according to the size and morphology of the tissue to meet different cell culture needs.

The thickness of the culture vessel 100 can be adjusted by controlling the amount of the mixed colloid. Different thicknesses will affect the performance and degree of deformation of the culture vessel 100. Thicker culture vessels 100 are simpler to operate and reduce the evaporation of the culture medium during the culture process. They are also less likely to be deformed and other problems. Therefore, when making the culture vessel 100, the appropriate thickness of the culture vessel 100 can be selected according to the experimental requirements and the convenience of operation. By adjusting the parameters of the culture vessel 100 according to the requirements of different tissues, a culture vessel 100 suitable for a specific experiment can be customized. These variable parameters can be flexibly adjusted according to the experimental requirements to ensure that the performance and use effect of the culture vessel 100 meet the goals and requirements of the research.

The present invention also relates to a cell culture method comprising the steps of:

a. Using the above-mentioned configuration device, first clean the culture vessel 100 and the culture box to remove impurities and residues on the surface of the culture vessel 100; for the culture vessel 100 made of polydimethylsiloxane, the culture vessel 100 can be cleaned with a phosphate buffered saline solution. The culture vessel 100 can be placed in the phosphate buffered saline solution and soaked for a preset time to remove impurities and residues on the surface of the culture vessel 100, and then the culture vessel 100 is placed in deionized water for ultrasonic cleaning to ensure the cleanliness of the culture dish surface.

b. Sterilize the incubator 100 and the culture box at high temperature to effectively kill any microorganisms remaining on the mold surface and reduce the risk of contamination. Remove the sterilized incubator 100 and culture box and gently wipe them dry with sterile filter paper to ensure the surface is dry. This operation can reduce bubbles generated in the culture tank during subsequent liquid addition.

For cells with special culture surface requirements, the inner wall of the culture box can be coated with a cell adhesive, which can be collagen or other cell adhesion substances to enhance cell attachment and growth;

c. Use sterile tweezers to place the culture vessel 100 in the culture box, which can be a culture dish or a well plate, ensuring that the culture vessel 100 fits perfectly with the inner wall of the well plate or culture dish and that there are no gaps or looseness;

d. Pipette the culture medium into the culture tank 1 from the input port. Specifically, use the pipette tip to accurately insert it into the bottom of the input port, ensuring that the tip is located inside the culture tank 1. Add the culture medium of pre-adjusted density from input port 2, paying attention to the volume added to ensure that the culture tank 1 is completely filled with culture medium.

When adding culture medium, be careful to remove air bubbles to ensure that the culture medium fills the culture tanks 1 and is in good contact with the cells. Slowly filling and gently shaking the incubator 100 can help remove air bubbles. Ensure that all culture tanks 1 are filled with culture medium and that the medium is evenly distributed.

By strictly following the aseptic operation process, correctly installing the culture vessel 100 and adding the culture medium containing the tissue, it is possible to ensure that the cells are cultured in good conditions in the culture tank 1. The implementation of these steps will help reduce contamination and cell damage, while improving the reliability and repeatability of the experiment.

In the process of cell culture, the ability of primary tissue to adhere to the wall is usually one of the key factors for the successful culture and expansion of cells. Many primary tissues, especially those derived from adult animals or humans, have the problem of poor adhesion. Traditional adherent culture methods cannot effectively separate and expand stem cells in tissues, and traditional primary cell anchoring methods (such as glass slide pressing) may cause tissue damage and cell inactivation. In addition, conventional methods of fixing tissues and primary cells may cause damage to tissue structure and cell integrity, affecting the accuracy of experimental results. In order to improve the repeatability and reliability of the experiment, we developed a culture vessel 100 to reduce damage to tissues and cells, and developed a cell culture method that enhances adhesion to better preserve the original characteristics of the tissue and improve the survival rate and proliferation ability of cells.

For the primary culture of stem cells, many factors need to be taken into account, such as extracellular matrix (ECM) components, cell-cell interactions, oxygen, nutrients, etc., to ensure that stem cells can maintain their characteristics and functions. The culture vessel 100 of the present invention can be provided with a plurality of small culture tanks 1, and the small culture tanks 1 can provide a culture environment with a smaller volume and closer to the original state, which helps to simulate the conditions in the body more accurately. According to the research of Bersini et al., small-volume culture devices can better simulate factors such as extracellular matrix components, cell-cell interactions, oxygen levels and nutrient supply, and promote more realistic experimental results and research findings. At the same time, for some primary tissues, especially human tissues with a small amount of tissue, traditional large-volume adherent culture still has many limitations in terms of tissue adhesion, culture medium volume, etc.

Traditional tissue fixation methods and conventional volume culture have many disadvantages. For example, slide-adherent culture requires the tissue sample to be pressed under the slide, which can easily cause mechanical trauma and death of cells, while hindering the exchange of nutrients and gases. In addition, this method requires a large amount of culture medium and a limited culture area, which increases costs and operational difficulties. In conventional volume culture, the culture medium is relatively large relative to the amount of tissue, resulting in sparse arrangement of tissue cells, which affects the interaction and communication between cells, especially for the proliferation and maintenance of stem cells.

In response to these problems, the present invention has developed a culture vessel 100 made of an elastic transparent material for fixing tissues and culturing primary cells. The device uses a small-volume culture tank 1 and breathable materials, which can reduce the risk of contamination and reduce the consumption of culture medium. The use of this device can reduce the mechanical trauma and mortality of cells, and provide microenvironmental conditions that are closer to the native state, which can improve the survival rate and proliferation rate of primary cells, and at the same time provide tissues with microenvironmental conditions that are closer to the native state. By optimizing the fixation and culture methods, the present invention is expected to improve the effect of tissue fixation and culture, and provide a more reliable experimental platform for cytological and biological research. In addition, the small culture tank 1 can also reduce the use of culture medium and containers, thereby reducing costs.

The following two experiments are designed to verify the cell culture effect of the culture vessel 100 of the present invention.

Experiment 1: Cultivating mouse brain tissue.

In primary tissue culture, tissue quantity is crucial for the success rate of primary culture. We first tested culture using readily available brain tissue to observe the effect of the Incubator 100 on primary cell culture, given the high success rate. We isolated neural progenitor cells (NPCs) from mouse hippocampal tissue within 24 hours of birth. These cells are abundant in the hippocampus and have a high primary culture success rate.

The following is the process of mouse sampling, tissue pretreatment and culture:

1. Prepare mouse NPC culture medium. The composition of the culture medium is shown in Figure 4a. Preparation of P0 newborn mice: Select pregnant mice codenamed C57BL/6J and obtain hippocampal tissue from the newborn mice within 24 hours after giving birth.

2. Newborn healthy mice were disinfected with 75% (volume fraction) alcohol and killed by cervical dislocation under sterile conditions. The scalp and skull were cut open, and the brain tissue was removed and placed in a dish containing 1% penicillin-streptomycin mixture in Dulbecco's phosphate buffered saline (DPBS buffer) (placed on ice).

3. Aseptically isolate the hippocampus. Keeping the dorsal surface of the brain facing upward, carefully flip open the cerebral cortex under a microscope to expose the hippocampus. Use ophthalmic scissors or sharp forceps to separate the tissue surrounding the hippocampus. Remove the hippocampus and place it in a dish containing #2 culture medium.

4. Use iris scissors to cut the tissue into pieces smaller than 1 ^mm3 , grind and filter the tissue using a sterile 200-mesh sieve, quantify the volume of the obtained tissue, and resuspend it in 2# culture medium to the working concentration.

5. Place the prepared sterile incubator 100 in a culture box using a 6-well plate. Ensure that there is no liquid in the culture tank of the incubator 100 and that the lower edge of the incubator 100 fits perfectly with the bottom wall of the hole without any gap. Add appropriate amounts of tissue into the culture tank 1, and set up a small amount of tissue culture group diluted 2 times and 5 times in the incubator 100, and a normal condition control group in the corresponding 24-well plate for comparative testing. In addition, three sizes of incubators 100 and 24-well plates were used as comparative experiments in this experiment. The depths of the culture tanks of the three sizes of incubators 100 were 0.2 mm, 0.5 mm, and 1 mm, respectively.

During the culture process, the cell culture medium was changed every three days to ensure that the cells had sufficient nutrients for growth.

Subculture and identification of NPCs

Figure 4b shows the cell growth observed on the fourth day of primary culture of mouse brain tissue in 0.2mm, 0.5mm, and 1mm culture troughs, as well as in a 24-well plate. As shown in Figure 4b, when the brain tissue inoculum was 2.5uL, a large number of cells grew in all three sizes of incubator 100 and the 24-well plate, representing the highest tissue concentration. When the brain tissue inoculum was 0.5uL, uneven cell growth and numerous gaps were observed in the 24-well plate, while cells in culture trough 1 of incubator 100 grew uniformly, with a confluence exceeding 80%. When the brain tissue inoculum was 0.25uL, poor cell growth was observed in the 24-well plate, with cells mostly scattered and few cells crawling out from around the tissue mass. Cells in the culture trough grew more than those surrounding the tissue mass, exhibiting a higher confluence than in the 24-well plate, and exhibited a more uniform morphology and even distribution.

Figure 5 is a summary comparison of the number of P0 cells in each group in three parallel repeated experiments. It can be seen that the 2.5ul tissue amount group obtained the most cells in the 24-well plate; however, when the tissue amount was drastically reduced, the 0.25ul tissue amount group, the 100 cells harvested in each culture vessel group were more than those in the 24-well plate group.

Figures 6a and 6b compare the cell yield per unit area and per unit volume of primary culture of mouse brain tissue. Further data analysis reveals that the trend in cell yield per unit area is similar to that of total cell yield (Figure 6a). However, per unit volume of culture medium, the low-volume culture tank protocol yields a higher cell yield, with the 0.25 μl tissue group exhibiting a difference of over 30-fold (Figure 6b).

The following is the sphere culture of mouse NPC to verify the cell purity.

The primary cultured cells were subcultured and adherently cultured for three consecutive passages to purify the cells and obtain relatively simple cells. The harvested cells were cultured in a 24-well tissue culture treated (TC treated) plate. Figure 7 shows the neurosphere culture on day 8. The results show that cells cultured in the incubator 100 and the 24-well plate can be cultured into neurospheres through suspension culture, demonstrating that the purity of NPCs is within an acceptable standard range and that the incubator 100 does not significantly affect the differentiation ability and characteristics of the cells.

In this part of the experiment, we compared cells cultured using culture vessel 100 with those cultured in traditional 24-well culture plates and found that culture vessel 100 showed obvious advantages at low tissue amounts and was able to obtain more cells. In addition, the use of culture vessel 100 promoted uniform cell growth, improved cell confluence, and made cell distribution more even. This is very important for maintaining cell status, reducing cell loss, and ensuring consistency of experiments. Another significant finding is that under the same volume, culture vessel 100 obtained more cells, which means that under the same experimental conditions, culture vessel 100 requires less culture medium, thereby saving experimental costs. In addition, we speculate that the use of culture vessel 100 may have created a better cell microenvironment and promoted cell proliferation.

Experiment 2: Dental Pulp Stem Cell Culture

Dental pulp mesenchymal stem cells are a type of multipotent stem cells present in human dental pulp tissue. They have high regenerative ability and multidirectional differentiation potential, and are therefore widely used in fields such as tissue engineering and regenerative medicine. Therefore, the present invention also uses dental pulp tissue to verify the effectiveness of the culture device 100.

Pre-culture device:

Preparation of the culture vessel 100: First, a custom-sized culture vessel 100 is prepared based on the characteristics of mouse dental pulp tissue. The vessel is then demolded and sterilized by autoclaving. Autoclaving effectively kills bacteria and viruses on the mold surface, reducing the risk of contamination during cell culture.

Pre-culture treatment: The sterilized culture vessel 100 is stored in pure water or phosphate buffered saline (PBS). The culture vessel 100 is taken out in advance and the surface moisture is absorbed with sterile filter paper, because water droplets will introduce bubbles when the culture medium is added later.

The following is the process of mouse dental pulp sampling, tissue pretreatment and culture:

Tissue cleaning and separation: The mice were killed by cervical dislocation, the heads were cut open and the lower incisors (including the mandible) of the mice were removed, and the surrounding tissues were carefully removed to reduce contamination from other tissues.

Mouse incisors were placed in PBS containing antibiotics diluted to a working concentration and rinsed multiple times to remove surface blood and contamination. Using a surgical knife, the bone at the root was carefully pried open along the tooth's shape to expose the dental pulp. The distal pulp tissue was carefully removed, retaining the root portion (approximately 1-2 mm) containing the most stem cells.

Collect enough tissue and transfer it to the cap of a 1.5ml centrifuge tube. Add about 10ul of complete culture medium (culture medium + 20% fetal bovine serum + 1% penicillin-streptomycin mixture). Use microscissors to mince the tissue as much as possible. Then add about 200ul of culture medium and collect it in a centrifuge tube. Store at 4°C or on ice until use.

Cell culture in culture vessel 100: Place the pre-dried culture vessel 100 in a culture dish, and add the isolated mouse dental pulp tissue suspension into the culture vessel 100 (the depth of the culture tank is 0.17 mm) and the 24 wells of the control at 30 ul/well. Be careful when adding the culture medium to prevent bubbles from appearing in the culture tank.

During the culture process, the culture medium should be replaced approximately every three days to provide sufficient nutrients to support cell proliferation and differentiation. The cells should also be observed and recorded. If the culture medium evaporates quickly, the cells can be placed in a humidified chamber, which can then be placed in an incubator.

Cells cultured in culture vessel 100 were compared to conventional adherent culture controls to evaluate the effectiveness of culture vessel 100 culturing.

Cell Digestion and Passaging: When cells have grown to a sufficient number, they need to be digested and passaged. During digestion, cells can be separated from the culture dish or incubator 100 using diluted trypsin or other digestive enzymes, and then transplanted to a new culture dish for the next round of culture. During cell passaging, it is important to control cell density and digestion time to ensure healthy cell growth and stability.

Passaging, culture and identification of dental pulp stem cells.

Multiple tissues and cell morphology in primary culture: After the tissue suspension is added to the 0.17 mm deep culture tank and 24-well plate of the culture vessel 100, the tissue fragments will settle to the bottom of the culture dish. Compared with the 24-well plate, the culture vessel 100 can better fix the tissue, thereby promoting the adherent stem cells to crawl out of the tissue. As shown in Figure 8, at different time points of culture, the cells in the culture vessel 100 crawled out faster and the clone size was significantly larger. On the second day, it was already possible to see cells in the tissue of the culture vessel 100 group begin to crawl out, and on the fifth day, more cells had proliferated.

On day 12 of culture, the total number of positive colonies was calculated. A comparison revealed that approximately 40% of the tissues in the culture tanks had grown into stem cell colonies, while the positive colony rate in the 24-well plates was only around 8%-25%, as shown in Figure 9A. When the clones proliferated to the point where they could be passaged (typically 10-13 days), the cells were digested and counted. The total yield in the culture tanks reached approximately 6 x ¹⁰⁴ / ^cm2 , as shown in Figure 9B, while the total yield in the 24-well plates was only 3 x ¹⁰⁴ / ^cm2 , a difference of up to 2-fold. Furthermore, in terms of relative volume, as shown in Figure 9C, the volume within the culture vessel 100 reached 1.08 x ¹⁰⁷ /ml, while that in the 24-well plates was only 2.8 x ¹⁰⁵ /ml, a difference of approximately 50-fold.

In order to further verify the effect of the culture vessel 100 on primary tissue, a comparison of 96-well and 0.2mm thick culture troughs was added. A piece of rat dental pulp tissue was added to each 96-well and 0.2mm thick culture trough to compare the crawling degree, ratio, and number of subsequent cells obtained of primary cells.

It can be clearly observed from the image in Figure 10 that, compared with the conventional 96-well adherent culture, the cells in the culture tank grow faster, in larger numbers, and with a larger clone area.

Figure 11 shows the culture conditions and cell crawling rate of dental pulp tissue in a 96-well plate and incubator 100. Figure 12 shows the cell counts of dental pulp tissue in a 96-well plate and incubator 100. Figures 13 and 14 show the cell crawling rate and cell harvest number in the well plate and incubator 100, respectively. Figure 15 shows the cell culture conditions in a 96-well plate and incubator 100.

On the sixth day after culture, the total number of positive clones was calculated and compared to find that stem cells had crawled out of the rat tissues in the 100-well culture device group, while the positive clone rate in the 96-well plate was only about 65.38%.

When clones proliferated to the point where they could be passaged, the cells were digested, counted, and cultured routinely. On day 6, the total tissue yield in the 100-well culture chamber reached approximately 4.16 x 10 ⁵ /cm ² , while the total tissue yield in the 96-well plate culture was 2.44 x 10 ⁵ /cm ² , a two-fold difference. Throughout the culture process, the cell morphology of the two groups was essentially similar.

After conventional culture at nearly the same culture concentration (2.1*10 ⁴ /cm2～2.2*10 ⁴ /cm ² ), the total cell yield of the 100-well culture device group reached 3.77*10 ⁷ on the 15th day, while the total cell yield of the 96-well plate culture group was 1.17*10 ⁷ , a difference of up to 3 times.

In primary culture experiments involving dental pulp, a small and challenging tissue, the Culture Device 100 demonstrated excellent performance, significantly outperforming traditional adherent controls in 24-well and 96-well plates in both colony and cell counts. It is speculated that the ability of Culture Device 100's culture chamber 1 to promote adherence to fixed tissue is the most important factor. Furthermore, the small culture volume of Culture Chamber 1 also significantly promotes the proliferation of primary cells.

It can be seen that for primary culture with difficult adherence and small tissue volume, such as dental pulp, the fixed culture in the culture tank 1 of the culture vessel 100 is a very good choice. Especially for clinical puncture samples or other tissues with limited quantity, this solution will greatly improve the success rate of primary culture.

While preferred embodiments of the present invention have been described in detail above, it should be understood that aspects of the embodiments can be modified, if necessary, to employ aspects, features and concepts of the various patents, applications and publications to provide further embodiments.

These and other changes can be made to the embodiments in light of the above detailed description.In general, in the claims, the terms used should not be construed as limited to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which these claims are entitled.

Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and that in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims (19)

A culture container, characterized in that the culture container is made of elastic transparent material and is provided with: a culture tank, the culture tank being formed by a depression in the bottom surface of the culture vessel; an input hole, the input hole being formed by the top wall of the culture tank being recessed into the top surface of the culture vessel; and An output hole is formed by the top wall of the culture tank being recessed to the top surface of the culture vessel and is spaced apart from the input hole. The incubator according to claim 1, wherein the plurality of culture tanks are respectively formed by depressions on the bottom surface of the incubator and are spaced apart; The plurality of input holes are respectively formed by the top walls of the plurality of culture tanks being recessed into the top surface of the culture vessel; The plurality of output holes are respectively formed by the top walls of the plurality of culture tanks being recessed into the top surface of the culture container and are respectively spaced apart from the plurality of corresponding input holes. The culture vessel according to claim 1 is characterized in that the culture vessel is made of polydimethylsiloxane. The culture vessel according to claim 1 is characterized in that the radial dimension of the output hole is larger than the radial dimension of the input hole. The culture device according to claim 1 is characterized in that the output hole diameter ranges from 1.5 mm to 2.5 mm, and the input hole diameter ranges from 0.5 mm to 1.5 mm. The culture vessel according to claim 1 is characterized in that the culture tank is long and narrow and its extension direction is perpendicular to the depth direction of the culture tank. A culture device, characterized in that the culture device comprises: a culture box having a receiving space and an open top; and The incubator according to claim 1 is located in the accommodating space, and the bottom surface of the incubator is in close contact with the bottom wall of the incubation box, and the side walls of the incubator are in close contact with the inner wall of the incubation box. The culture device according to claim 7 is characterized in that the culture vessel is a cylindrical structure and the culture box is a culture dish or a multi-well plate. The culture device according to claim 8 is characterized in that the thickness of the culture vessel ranges from 0.5 cm to 1 cm, and the depth of the culture tank ranges from 0.1 mm to 5 mm. The culture device according to claim 8, characterized in that the height of the culture vessel is less than the depth of the culture box. A method for processing a culture vessel, characterized in that it comprises the steps of: S1. Mixing the silicon polymer and the cross-linking agent in proportion and stirring evenly to form a mixed colloid; S2, extracting bubbles in the mixed colloid; S3, stacking the sheet-shaped mold on the bottom wall of the culture box, then pouring the mixed colloid after the bubbles are extracted into the culture box, and heating and drying until the mixed colloid is solidified to form a culture container; S4. After taking out the culture vessel, the mold is separated from the culture vessel, and a culture tank is formed on the bottom surface of the culture vessel; an input hole and an output hole are punched on the top wall of the culture tank with a puncher, and the input hole and the output hole pass through the culture vessel respectively. The processing method of the culture vessel according to claim 11 is characterized in that the culture box is a culture dish and the mold is a glass slide. The method for processing an incubator according to claim 12, wherein in step S2, a plurality of the molds are respectively stacked on the bottom wall of the incubator box and spaced apart; In step S4, the plurality of molds are separated from the culture vessel to form a plurality of culture tanks, and the input holes and the output holes are punched out on the top walls of the plurality of culture tanks using a puncher. The processing method of the culture vessel according to claim 12 is characterized in that at least two of the molds are stacked in the vertical direction. The processing method of the culture vessel according to claim 14 is characterized in that the sizes of at least two of the molds are different. The processing method of the culture vessel according to claim 11 is characterized in that, in step S3, the heating temperature is 70°-80° and the time is 60min-70min. A cell culture method, characterized in that it comprises the steps of: a. Cleaning the culture device according to claim 11 to remove impurities and residues on the surface of the culture device; b. sterilizing the incubator and the culture box; c. placing the incubator in the culture box; d. Move the culture medium from the input port into the culture tank. The cell culture method according to claim 17, characterized in that the culture vessel is made of polydimethylsiloxane; in step a, the culture vessel is placed in a phosphate buffered saline solution and soaked for a preset time to remove impurities and residues on the surface of the culture vessel, and then the culture vessel is placed in deionized water for ultrasonic cleaning to ensure the cleanliness of the culture dish surface. The cell culture method according to claim 18, characterized in that, in step b, after the culture vessel is sterilized, the culture vessel is further wiped dry using sterile filter paper.