A manufacturing method of a 3D cell spheroid formation container and a culture container

Abstract

The application discloses a cell culture container, and aims to provide a manufacturing method of a 3D cell sphere forming container and the culture container with a surface having super-high flatness, good light transmittance and stable coating performance, and the technical scheme is that a choline phosphate coating liquid is evenly coated on the surface of a container base, so that cells can form regular spheres on the surface of the container base, the culture container after coating has high light transmittance, and the interference of optical imaging is reduced; under the synergistic effect of the choline phosphate ultra-low adsorption coating, the cells naturally settle and gather in the micro-cavity to form regular spheres, the coating on the surface of the culture container is a biocompatible choline phosphate polymer, the surface of the culture container has ultra-low adsorption characteristics, the cells cannot adhere to the surface and are forced to gather, and the finished culture container has excellent cell cultivation and observation effects, and the application is suitable for the field of cell culture container production.

Description

Technical Field

[0001] This invention relates to a cell culture container, and more specifically, to a method for manufacturing a 3D cell spheroid forming container and a culture container. Background Technology

[0002] 3D cell spheroid culture technology can effectively simulate the three-dimensional microenvironment of cell growth in the human body, realistically replicating biological behaviors such as cell proliferation, differentiation, intercellular interactions, and drug tolerance. It is a core in vitro research model in fields such as regenerative medicine, tumor pharmacology screening, organoid construction, and drug toxicity evaluation. However, existing containers and their preparation processes for 3D cell spheroid culture generally suffer from structural and technical defects, including poor spheroid uniformity, significant optical imaging interference, insufficient experimental reproducibility, and limited adaptability. These issues severely restrict the construction of high-precision 3D cell models and their large-scale application in high-throughput drug screening.

[0003] In existing technologies, the mainstream methods for preparing 3D cell spheroids mainly include three categories: matrix gel encapsulation culture, modified ultra-low adsorption container culture, and culture in dedicated equipment systems. All of these methods have significant technical drawbacks. Among them, matrix gel encapsulation culture relies on a gel matrix to achieve three-dimensional cell encapsulation, but the uneven distribution of pores within the matrix gel easily leads to obstructed oxygen and nutrient transport and accumulation of metabolic waste within the culture system. This results in hypoxic necrosis at the center of the cell spheroids, large dispersion in spheroid size and morphology, and poor batch-to-batch consistency. Furthermore, matrix gels themselves have poor light transmittance and are subject to autofluorescence interference, failing to meet the requirements for high-magnification microscopic imaging, dynamic real-time observation, and quantitative analysis. Moreover, the high material cost and complex operation procedures make them unsuitable for high-throughput screening scenarios.

[0004] Conventional commercial ultra-low adsorption culture containers are mostly prepared using polymer surface hydrophobic modification or plasma roughening processes. These processes increase the hydrophobicity of the container surface to inhibit cell adhesion and induce cell self-aggregation into spheroids. However, these processes have poor control over surface roughness, resulting in insufficient microscopic smoothness of the container's inner wall after molding. This easily leads to uneven cell aggregation, resulting in spheroids of varying sizes and irregular shapes, significantly reducing the accuracy and reliability of experimental data. Furthermore, the roughened structure easily induces optical refraction, imaging ghosting, and edge distortion, making it impossible to achieve high-definition microscopic imaging and precise quantitative analysis of spheroid size. In addition, traditional modified coatings have weak adhesion to the substrate; during culture, repeated medium changes and immersion in the culture medium can easily lead to localized coating detachment and failure, causing localized cell adhesion, spheroid disintegration, and poor system culture stability. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for manufacturing a 3D cell spheroid forming container and a culture container with ultra-high surface flatness, good light transmittance and stable coating performance.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for manufacturing a 3D cell spheroid forming container, comprising the following steps: S1, container substrate cleaning treatment: selecting conventional polystyrene culture plates, culture dishes, and matrix plates as substrates, and performing ultrasonic cleaning with sterile deionized water and ethanol solution to remove residual impurities on the substrate surface; S2. Container substrate drying treatment: The cleaned container substrate is placed in a sterile and dust-free oven for low-temperature drying, and the substrate surface is activated by low-temperature plasma to form a culture container. After the treatment, it is necessary to ensure the flatness of the substrate and that there are no scratches or protrusions on the surface. S3. Precise preparation of phosphoric acid choline coating solution: Biocompatible phosphoric acid choline polymers are selected as the core functional raw materials, and combined with anhydrous ethanol and sterile buffer solution to prepare a homogeneous and transparent functional coating solution. S4. High-precision flat coating: Through a precision dip coating process, the surface of the pretreated culture container is fully and uniformly coated to form a liquid film of uniform thickness on the surface of the culture container. S5. Drying and curing molding process: The coated culture container is placed in a dust-free environment simulation device with constant temperature and humidity. First, the temperature in the device is adjusted to 37℃, and the liquid film is leveled and shaped by a slow shaking process on an ultra-low speed platform. After the shaping is completed, the temperature is gradually increased to 50℃-70℃ within a period of 1-2 hours to achieve full curing of polymer molecules on the surface of the culture container. S6. Post-processing and quality control testing: After curing, the culture container is sterilized with ethylene oxide. At the same time, the surface flatness, light transmittance, and cell ultra-low adsorption of the culture container are tested. If the culture container passes the test, it is shipped and stored. Otherwise, the culture container is reworked based on the unqualified performance.

[0007] The present invention is further configured such that step S3 also includes a method for preparing the coating liquid, comprising the following steps: S31, dissolving the biocompatible phosphoric acid choline polymer in anhydrous ethanol to form a stock solution of 8 mg / mL; S32. Allow the stock solution to stand so that the biocompatible phosphoric acid choline polymers are completely dissolved in anhydrous ethanol. S33. After dissolution, dilute the stock solution to 1.6 mg / mL with sterile buffer and anhydrous ethanol, with the volume ratio of anhydrous ethanol to buffer being 1:1. S34. Allow the diluted stock solution to stand to ensure that the added anhydrous ethanol and buffer solution are fully dissolved.

[0008] The present invention is further configured such that step S4 includes the following steps: S41, immersing the processed culture container into the prepared stock solution at a uniform speed without vibration; S42. After the culture container is immersed, keep it still in the stock solution to allow the coating solution to fully wet the substrate surface; S43. Based on the required coating thickness, pull it out of the reservoir at a constant speed of 1cm / min-30cm / min without vibration.

[0009] The present invention is further configured such that step S5 includes the following steps: S51, placing the coated culture container on a constant temperature and humidity shaker at 37°C and keeping it horizontal to ensure that the liquid film on the surface of the culture container is evenly distributed under the action of gravity. S52. Start the shaker to start the shaker at an ultra-low speed and shake slowly, so that the coating liquid flows slowly and continuously on the substrate surface to eliminate local thickness unevenness, and run continuously for 13 hours. S53. After the low-temperature slow shaking process is completed, the processed culture container is transferred to a constant temperature dust-free oven, heated to 50°C at a rate of 1°C / min and maintained for 30 min, then heated to 70°C at a rate of 0.5°C / min and maintained for 60 min. S54. After curing, allow the coating to cool naturally to room temperature to prevent stress cracking.

[0010] The present invention is further configured such that step S6 includes the following steps: S61, placing the packaged culture container inside the sterilizer to ensure that ethylene oxide, heat and humidity can fully penetrate all products; S62. A biological indicator is installed inside the sterilization cabinet to detect the sterilization effect; S63. During the sterilization process, various parameters inside the sterilization cabinet are monitored to obtain the sterilization time, pressure, humidity, temperature, and ethylene oxide consumption inside the sterilization cabinet. S64. After sterilization, transfer the processed product to the testing equipment and perform forced ventilation analysis on the testing equipment for a duration of t. S65. After time period t, the residual amount of ethylene oxide in the detection equipment is detected, and the detection value is X. If X > 10 μg / g, it is determined that the current residual amount of ethylene oxide is too high, and ventilation is continued. Otherwise, it is determined that the current residual amount of ethylene oxide in the culture container is low, and the process jumps to S66 to perform physical performance testing. S66. Linearly scan the surface of the culture container using a profilometer to obtain the surface profile of the culture container. At the same time, inspect the surface profile. If no particles or cracks appear on the surface of the culture container, the surface flatness of the current culture container is deemed to be qualified, and proceed to S67 to continue the inspection. Otherwise, re-coat the surface of the culture container. S67. Using uncoated containers of the same material as a blank control group, and setting the transmittance of the control group to 100%, the culture containers after coating are tested for visible light, and the transmittance of the culture containers is judged. If the transmittance of the culture container is ≥95% relative to the transmittance of the control group, the transmittance of the current culture container is judged to be qualified, and the test is skipped to S68. Otherwise, the surface of the culture container is recoated. S68. The cells to be cultured are seeded at an appropriate density inside a coated culture container, cultured with suitable environmental parameters, and the morphology of the cells is detected. If the cells are suspended in a tightly packed spherical aggregate in the culture container, the current culture container is judged to have good adsorption. Conversely, if the giant cells are attached to the wall and spread out or loosely aggregated, the current culture container is judged to have poor adsorption.

[0011] Preferably, step S66 further includes surface contour flatness detection, including a protruding particle detection step: S661, on the surface of the culture container, a plurality of projection light paths are provided parallel to the surface of the culture container, one end of the projection light path is a light outlet, and the other end is a signal receiving port; S662. The projection optical path is suspended above the surface of the culture container to be tested and moves along the direction of the culture container to be tested. During the movement, the signal receiving port receives the optical signal. S663. If all of the signal receiving ports lose light signals at the same time, it is determined that no protruding particles appear on the surface of the current culture container. Conversely, if one or more of the signal receiving ports lose light signals, it is determined that there are protrusions on the surface of the culture container corresponding to the projection light path. And the crack detection steps: S664, set the light outlet at a position perpendicular to the surface of the culture container to be tested, with the light outlet facing the direction of the culture container and projecting a mesh detection light beam in the direction of the culture container; S665. An image acquisition device is installed on the light outlet. During the projection of light, the image acquisition device acquires the light information on the surface of the culture container. S666. If the light on the surface of the culture container is distorted or deflected, it is determined that the culture container at the current position has a dent or crack; otherwise, it is determined that the surface of the current culture container is flat.

[0012] This application also discloses a method for manufacturing a 3D cell spheroid forming container, the culture container including a mounting box and a sealing cover plate disposed above the mounting box. The mounting box is uniformly provided with a plurality of culture chambers along its length, and each culture chamber is provided with a plurality of microcavities at its bottom. The cells settle and aggregate within the enclosure to form spheroids.

[0013] The beneficial effects of adopting the above technical solution are as follows: 1. This application provides a method for manufacturing a 3D cell spheroid formation container. By smoothly coating the surface of the container substrate with a phosphoric acid choline coating solution, cells can form regular spheroids on the surface of the container substrate. Furthermore, the culture container after coating has high light transmittance, reducing interference with optical imaging. In terms of container structure, this application provides several culture chambers along its length within the mounting box. Each culture chamber has several microcavities at its bottom. Cells naturally settle and aggregate in the microcavities to form regular spheroids under the synergistic effect of the phosphoric acid choline ultra-low adsorption coating. Moreover, the coating on the surface of the culture container is a biocompatible phosphoric acid choline coating. Choline-based polymers enable the culture container surface to have ultra-low adsorption properties, preventing cells from adhering to the wall and forcing them to aggregate. At the same time, the coating has strong adhesion to the substrate and high stability, and does not detach or fail even after multiple medium changes and long-term culture. Furthermore, the container is dried and cured after coating, making the surface roughness of the coating close to 0, while retaining the high light transmittance of the substrate. This fundamentally eliminates problems such as optical refraction and image ghosting caused by low surface flatness of the culture container. After the culture container is manufactured, surface gloss, light transmittance, and ultra-low cell adsorption performance are tested to ensure that the physical properties, optical properties, and biological functions of the finished product all meet the preset standards.

[0014] 2. Furthermore, this application utilizes a precise preparation of a phosphocholine coating solution to form a homogeneous and transparent coating solution. During the preparation process, an 8 mg / mL stock solution is pre-prepared and allowed to stand until completely dissolved. Then, sterile buffer and anhydrous ethanol are added at a 1:1 volume ratio to dilute to 1.6 mg / mL, and the solution is allowed to stand again until fully dissolved. This multi-step preparation method ensures that the polymer molecules in the coating solution are fully extended and uniformly dispersed, avoiding microscopic defects in the coating caused by polymer agglomeration and insufficient dissolution in traditional simple preparation methods. Simultaneously, to ensure uniform thickness on the surface of the culture container, a precise dip-coating process is used. The culture container is pushed into the coating solution at a uniform speed without vibration, allowing the coating solution to fully wet the substrate surface. The culture container is then uniformly lifted out again. The coating thickness can be precisely controlled by adjusting the lifting speed; the faster the lifting speed, the thinner the coating thickness. Furthermore, the placement and removal of the culture container are ensured to be uniform and vibration-free. The liquid film is guaranteed to flow and level autonomously on the substrate surface, forming an ultra-thin liquid film without pinholes, wrinkles, or uneven thickness. Furthermore, the amphipathic phosphocholine molecules can adsorb 7-8 water molecules to form a water wall, preventing cells, proteins, and other substances from adhering to the container surface, thus achieving excellent ultra-low adsorption performance. At the same time, the MPC polymer has good adhesion to the synthetic resin, ensuring the long-term stability of the coating. The organic combination of the above formulation and coating process makes the coating uniformity, thickness controllability, and ultra-low adsorption performance of the culture container of the present invention significantly superior to the prior art, providing a reliable structural guarantee for the uniform formation of cell spheroids.

[0015] 3. Simultaneously, during the drying and curing process of the coating liquid, this application employs a staged gradient drying and curing process to ensure both the full drying and curing of the coating and the optical performance of the culture container. During the drying and curing process, the coated culture container is first placed in a constant temperature and humidity environment at 37°C and slowly shaken on an ultra-low speed platform to achieve liquid film leveling and shaping. By extending the leveling window period, the coating liquid is allowed to fully and autonomously level on the substrate surface, eliminating any minor thickness unevenness that may occur during the coating process. Then, the temperature is gradually increased to 50°C at a rate of 1°C / min and maintained for 30 minutes, followed by a gradual increase at 0.5°C / min. The temperature is gradually increased to 70°C and maintained for 60 minutes. This gradient heating avoids stress cracking of the coating caused by thermal shock, while ensuring that the polymer molecular chains receive sufficient thermal energy for rearrangement and cross-linking, forming a dense and stable functional layer. After curing, the coating is naturally cooled to room temperature to further prevent stress cracking. The cured culture container is then sterilized with ethylene oxide at a temperature lower than the thermal decomposition temperature of the phosphocholine polymer. This ensures sterility without causing thermal damage to the coating structure or the container substrate. After sterilization, the product is subjected to forced ventilation desorption until the residual ethylene oxide concentration X ≤ 10 μg / mL, ensuring the product's biocompatibility. The synergistic effect of the gradient drying curing and sterilization processes achieves an optimal balance between coating density, substrate adhesion, and biocompatibility in the culture container described in this invention.

[0016] 4. Furthermore, this application establishes a triple quality control system of "full inspection of surface smoothness—full inspection of transmittance—batch sampling inspection of ultra-low cell adsorption performance," and combines it with an optimized microcavity structure design for the culture container. This achieves comprehensive quality control from physical performance to optical performance and biological function. Regarding surface smoothness detection, this invention uses a profilometer to perform linear scanning along the surface of the culture container, acquiring the surface profile and detecting the presence of particles or cracks, thus fundamentally avoiding the problem of uneven cell aggregation caused by traditional roughening processes. Regarding transmittance detection, this invention uses an uncoated container of the same material as a blank control group (transmittance set to 100%) to detect the transmittance of the coated culture container in the visible light range. The transmittance of the culture container is required to be ≥95% relative to the control group, ensuring the optical applicability of the culture container in high-magnification microscopic imaging, dynamic real-time observation, and quantitative analysis scenarios. Regarding the testing of ultra-low adsorption performance of cells, this invention involves seeding cells at an appropriate density into a coated culture container and culturing them. The ultra-low adsorption performance of the coating is determined by observing the cell morphology—if the cells are suspended and tightly aggregated into spherical shapes within the culture container, the adsorption performance is deemed acceptable; conversely, if the cells adhere to the wall and spread out or loosely aggregate, the adsorption performance is deemed poor. The culture container of this application includes a mounting box and a sealing cover. Several culture chambers are uniformly arranged along the length of the mounting box, and several microcavities are provided at the bottom of each culture chamber. Cells naturally settle and aggregate in the microcavities to form spheres. The synergistic effect of the triple quality control system and the microcavity structure of the container ensures that each batch of products meets the preset standards in the three core indicators of surface smoothness, light transmittance, and ultra-low adsorption performance of cells. This provides a full-process quality assurance for the uniformity of 3D cell sphere formation, high-definition imaging, and high reproducibility of experimental data.

[0017] 5. Furthermore, to further improve cell aggregation and observability within the culture container, it is necessary to ensure the surface of the culture container is smooth after the coating solution is applied. This prevents the formation of pits, cracks, or raised particles, which would hinder cell aggregation and the formation of spherical masses, thus affecting observation results. Therefore, after the coating solution is applied, the smoothness of the culture container surface must be ensured. Surface protrusions are detected by a projection light path along the surface to be tested. This projection light path is generated through a light outlet, and the trajectory of the light outlet matches the cross-sectional trajectory of the surface to be tested on the culture container. The projection light path moves towards the surface to be tested. If a protrusion exists on the surface, it will be detected as the projection light path moves... During the process, the projection light path corresponding to the protrusion position will be blocked in advance and detected by the signal receiving port, thereby determining that there is a protruding particle at the current position. Conversely, if all the signal receiving ports lose signals at the same time, it is determined that the light output port is blocked by the culture container, and there is no protrusion on the surface of the culture container. Similarly, by setting a light output port on the surface to be tested of the culture container, the mesh detection light is projected perpendicularly to the surface to be tested of the culture container. If there are depressions or cracks on the surface, the light projected at the cracks will be distorted and deflected. By setting an image acquisition device to collect and detect the mesh detection light, it is possible to conveniently obtain the flatness data of the culture container surface and prevent the depressions and protrusions on the culture container surface from affecting the cell culture effect. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the fabrication method of a 3D cell spheroid forming container and an embodiment of the culture container of the present invention. Figure 2 This is a flowchart illustrating the preparation method of the coating liquid in an embodiment of a method for manufacturing a 3D cell spheroid forming container and a culture container according to the present invention. Figure 3 This is a flowchart of a method for fabricating a 3D cell spheroid forming container and a method for coating the surface of a culture container according to an embodiment of the present invention. Figure 4 This is a flowchart of a drying and curing molding method for a method of fabricating a 3D cell spheroid forming container and an embodiment of a culture container according to the present invention; Figure 5 This is a flowchart of a method for fabricating a 3D cell spheroid forming container and a post-processing and quality control method for an embodiment of the culture container of the present invention. Figure 6 This is a flowchart illustrating a method for fabricating a 3D cell spheroid forming container and a surface contour flatness detection method for an embodiment of the present invention. Detailed Implementation

[0019] Reference Figures 1 to 6 The present invention provides a method for fabricating a 3D cell spheroid forming container and an embodiment of the culture container.

[0020] For ease of explanation, spatial relative terms such as “up,” “down,” “left,” and “right” are used in the embodiments to describe the relationship of one element or feature shown in the figures relative to another element or feature. It should be understood that, in addition to the orientations shown in the figures, spatial terms are intended to include different orientations of the device in use or operation. For example, if the device in the figures is inverted, an element described as being “down” of other elements or features would be positioned “up” of those other elements or features. Therefore, the exemplary term “down” can encompass both up and down orientations. The device may be positioned in other ways (rotated 90 degrees or in other orientations), and the spatial relative descriptions used herein will be interpreted accordingly.

[0021] Moreover, relational terms such as “first” and “second” are used merely to distinguish one component from another that has the same name, without necessarily requiring or implying any such actual relationship or order between the components.

[0022] A method for fabricating a 3D cell spheroid formation container includes the following steps: S1, container substrate cleaning treatment: Select a culture plate, culture dish, or matrix plate made of conventional polystyrene material as the substrate, and perform ultrasonic cleaning with sterile deionized water and ethanol solution to remove residual impurities on the substrate surface; S2. Container substrate drying treatment: The cleaned container substrate is placed in a sterile and dust-free oven for low-temperature drying, and the substrate surface is activated by low-temperature plasma to form a culture container. After the treatment, it is necessary to ensure the flatness of the substrate and that there are no scratches or protrusions on the surface. S3. Precise preparation of phosphoric acid choline coating solution: Biocompatible phosphoric acid choline polymers are selected as the core functional raw materials, and combined with anhydrous ethanol and sterile buffer solution to prepare a homogeneous and transparent functional coating solution. S4. High-precision flat coating: Through a precision dip coating process, the surface of the pretreated culture container is fully and uniformly coated to form a liquid film of uniform thickness on the surface of the culture container. S5. Drying and curing molding process: The coated culture container is placed in a dust-free environment simulation device with constant temperature and humidity. First, the temperature in the device is adjusted to 37℃, and the liquid film is leveled and shaped by a slow shaking process on an ultra-low speed platform. After the shaping is completed, the temperature is gradually increased to 50℃-70℃ within a period of 1-2 hours to achieve full curing of polymer molecules on the surface of the culture container. S6. Post-processing and quality control testing: After curing, the culture container is sterilized with ethylene oxide. At the same time, the surface flatness, light transmittance, and cell ultra-low adsorption of the culture container are tested. If the culture container passes the test, it is shipped and stored. Otherwise, the culture container is reworked based on the unqualified performance.

[0023] Step S3 further includes a method for preparing the coating solution, comprising the following steps: S31, dissolving the biocompatible phosphoric acid choline polymer in anhydrous ethanol to form an 8 mg / mL stock solution; S32. Allow the stock solution to stand so that the biocompatible phosphoric acid choline polymers are completely dissolved in anhydrous ethanol. S33. After dissolution, dilute the stock solution to 1.6 mg / mL with sterile buffer and anhydrous ethanol, with the volume ratio of anhydrous ethanol to buffer being 1:1. S34. Allow the diluted stock solution to stand to ensure that the added anhydrous ethanol and buffer solution are fully dissolved.

[0024] Step S4 further includes the following steps: S41, immersing the processed culture container into the prepared stock solution at a uniform speed without vibration; S42. After the culture container is immersed, keep it still in the stock solution to allow the coating solution to fully wet the substrate surface; S43. Based on the required coating thickness, pull it out of the reservoir at a constant speed of 1cm / min-30cm / min without vibration.

[0025] Step S5 further includes the following steps: S51, In an environment of 37°C, the coated culture container is placed on a constant temperature and humidity shaker and kept horizontal to ensure that the liquid film on the surface of the culture container is evenly distributed under the action of gravity. S52. Start the shaker to start the shaker at an ultra-low speed and shake slowly, so that the coating liquid flows slowly and continuously on the substrate surface to eliminate local thickness unevenness, and run continuously for 13 hours. S53. After the low-temperature slow shaking process is completed, the processed culture container is transferred to a constant temperature dust-free oven, heated to 50°C at a rate of 1°C / min and maintained for 30 min, then heated to 70°C at a rate of 0.5°C / min and maintained for 60 min. S54. After curing, allow the coating to cool naturally to room temperature to prevent stress cracking.

[0026] Step S6 further includes the following steps: S61, placing the packaged culture containers inside the sterilizer to ensure that ethylene oxide, heat and humidity can fully penetrate all products; S62. A biological indicator is installed inside the sterilization cabinet to detect the sterilization effect; S63. During the sterilization process, various parameters inside the sterilization cabinet are monitored to obtain the sterilization time, pressure, humidity, temperature, and ethylene oxide consumption inside the sterilization cabinet. S64. After sterilization, transfer the processed product to the testing equipment and perform forced ventilation analysis on the testing equipment for a duration of t. S65. After time period t, the residual amount of ethylene oxide in the detection equipment is detected, and the detection value is X. If X > 10 μg / g, it is determined that the current residual amount of ethylene oxide is too high, and ventilation is continued. Otherwise, it is determined that the current residual amount of ethylene oxide in the culture container is low, and the process jumps to S66 to perform physical performance testing. S66. Linearly scan the surface of the culture container using a profilometer to obtain the surface profile of the culture container. At the same time, inspect the surface profile. If no particles or cracks appear on the surface of the culture container, the surface flatness of the current culture container is deemed to be qualified, and proceed to S67 to continue the inspection. Otherwise, re-coat the surface of the culture container. S67. Using uncoated containers of the same material as a blank control group, and setting the transmittance of the control group to 100%, the culture containers after coating are tested for visible light, and the transmittance of the culture containers is judged. If the transmittance of the culture container is ≥95% relative to the transmittance of the control group, the transmittance of the current culture container is judged to be qualified, and the test is skipped to S68. Otherwise, the surface of the culture container is recoated. S68. The cells to be cultured are seeded at an appropriate density inside a coated culture container, cultured with suitable environmental parameters, and the morphology of the cells is detected. If the cells are suspended in a tightly packed spherical aggregate in the culture container, the current culture container is judged to have good adsorption. Conversely, if the giant cells are attached to the wall and spread out or loosely aggregated, the current culture container is judged to have poor adsorption.

[0027] Preferably, step S66 further includes surface contour flatness detection, including a protruding particle detection step: S661, on the surface of the culture container, a plurality of projection light paths are provided parallel to the surface of the culture container, one end of the projection light path is a light outlet, and the other end is a signal receiving port; S662. The projection optical path is suspended above the surface of the culture container to be tested and moves along the direction of the culture container to be tested. During the movement, the signal receiving port receives the optical signal. S663. If all of the signal receiving ports lose light signals at the same time, it is determined that no protruding particles appear on the surface of the current culture container. Conversely, if one or more of the signal receiving ports lose light signals, it is determined that there are protrusions on the surface of the culture container corresponding to the projection light path. And the crack detection steps: S664, set the light outlet at a position perpendicular to the surface of the culture container to be tested, with the light outlet facing the direction of the culture container and projecting a mesh detection light beam in the direction of the culture container; S665. An image acquisition device is installed on the light outlet. During the projection of light, the image acquisition device acquires the light information on the surface of the culture container. S666. If the light on the surface of the culture container is distorted or deflected, it is determined that the culture container at the current position has a dent or crack; otherwise, it is determined that the surface of the current culture container is flat.

[0028] This application also discloses a method for manufacturing a 3D cell spheroid forming container, the culture container including a mounting box and a sealing cover plate disposed above the mounting box. The mounting box is uniformly provided with a plurality of culture chambers along its length, and each culture chamber is provided with a plurality of microcavities at its bottom. The cells settle and aggregate within the enclosure to form spheroids.

[0029] This application provides a method for fabricating a 3D cell spheroid formation container. By smoothly coating the surface of the container substrate with a phosphoric acid choline coating solution, cells can form regular spheroids on the container substrate surface. Furthermore, the coated culture container has high light transmittance, reducing interference with optical imaging. At the container structure level, this application provides several culture chambers along the length of the mounting box, and each culture chamber has several microcavities at its bottom. Cells naturally settle and aggregate within the microcavities to form regular spheroids under the synergistic effect of the phosphoric acid choline ultra-low adsorption coating. The coating on the surface of the culture container is a biocompatible phosphoric acid choline polymer, capable of… This process gives the culture container surface ultra-low adsorption properties, preventing cells from adhering to the wall and forcing them to aggregate. At the same time, the coating has strong adhesion to the substrate and high stability, and does not detach or fail even after multiple medium changes and long-term culture. Furthermore, the container is dried and cured after coating, making the surface roughness of the coating close to 0, while retaining the high light transmittance of the substrate. This fundamentally eliminates problems such as optical refraction and image ghosting caused by low surface flatness of the culture container. After the culture container is manufactured, the surface gloss, light transmittance, and ultra-low adsorption performance of the cells are tested to ensure that the physical properties, optical properties, and biological functions of the finished product all meet the preset standards.

[0030] Furthermore, this application employs a precise preparation of a phosphocholine coating solution to form a homogeneous and transparent coating solution. During preparation, an 8 mg / mL stock solution is pre-prepared and allowed to stand until completely dissolved. Then, sterile buffer and anhydrous ethanol are added at a 1:1 volume ratio to dilute to 1.6 mg / mL, and the solution is allowed to stand again until fully dissolved. This multi-step preparation method ensures that the polymer molecules in the coating solution are fully extended and uniformly dispersed, avoiding microscopic defects in the coating caused by polymer agglomeration and incomplete dissolution in traditional simple preparation methods. Simultaneously, to ensure uniform thickness on the surface of the culture container, a precise dip-coating process is used. The culture container is pushed into the coating solution at a uniform speed without vibration, allowing the coating solution to fully wet the substrate surface. The culture container is then uniformly lifted out again. The coating thickness can be precisely controlled by adjusting the lifting speed; the faster the lifting speed, the thinner the coating thickness. Furthermore, the placement and removal of the culture container are ensured to be uniform and vibration-free. The liquid film is guaranteed to flow and level autonomously on the substrate surface, forming an ultra-thin liquid film without pinholes, wrinkles, or uneven thickness. Furthermore, the amphipathic phosphocholine molecules can adsorb 7-8 water molecules to form a water wall, preventing cells, proteins, and other substances from adhering to the container surface, thus achieving excellent ultra-low adsorption performance. At the same time, the MPC polymer has good adhesion to the synthetic resin, ensuring the long-term stability of the coating. The organic combination of the above formulation and coating process makes the coating uniformity, thickness controllability, and ultra-low adsorption performance of the culture container of the present invention significantly superior to the prior art, providing a reliable structural guarantee for the uniform formation of cell spheroids.

[0031] Meanwhile, during the drying and curing process of the coating liquid, this application employs a staged gradient drying and curing process to ensure both the full drying and curing of the coating and the optical performance of the culture container. In the drying and curing process, the coated culture container is first placed in a constant temperature and humidity environment at 37°C and slowly shaken on an ultra-low speed platform to achieve liquid film leveling and shaping. By extending the leveling window period, the coating liquid is allowed to fully and autonomously level on the substrate surface, eliminating any minor thickness unevenness that may occur during the coating process. Then, the temperature is gradually increased to 50°C at a rate of 1°C / min and maintained for 30 minutes, followed by a further increase at 0.5°C / min. The temperature is raised to 70°C and maintained for 60 minutes. This gradient heating avoids stress cracking of the coating due to thermal shock, while ensuring the polymer molecular chains receive sufficient thermal energy for rearrangement and cross-linking, forming a dense and stable functional layer. After curing, natural cooling to room temperature further prevents stress cracking. The cured culture container is then sterilized with ethylene oxide at a temperature lower than the thermal decomposition temperature of the phosphocholine polymer. This ensures sterility without causing thermal damage to the coating structure or the container substrate. After sterilization, forced ventilation is used to desorb the product until the residual ethylene oxide level X ≤ 10 μg / mL, ensuring the product's biosafety. The synergistic effect of the gradient drying curing and sterilization processes achieves an optimal balance between coating density, substrate adhesion, and biosafety in the culture container described in this invention.

[0032] Furthermore, this application establishes a triple quality control system of "full inspection of surface smoothness, full inspection of transmittance, and batch sampling inspection of ultra-low cell adsorption performance," combined with an optimized microcavity structure design for the culture container, achieving comprehensive quality control from physical properties to optical properties and biological functions. Regarding surface smoothness detection, this invention uses a profilometer to perform linear scanning along the surface of the culture container, acquiring the surface profile and detecting the presence of particles or cracks, fundamentally avoiding the problem of uneven cell aggregation caused by traditional roughening processes. Regarding transmittance detection, this invention uses an uncoated container of the same material as a blank control group (transmittance set to 100%) to detect the transmittance of the coated culture container in the visible light range, requiring the transmittance of the culture container to be ≥95% relative to the control group, ensuring the optical applicability of the culture container in high-magnification microscopic imaging, dynamic real-time observation, and quantitative analysis scenarios. Regarding the detection of ultra-low adsorption performance of cells, this invention involves seeding cells at an appropriate density into a coated culture container and culturing them. The ultra-low adsorption performance of the coating is determined by observing the cell morphology. If the cells are suspended and tightly aggregated into spherical shapes within the culture container, the adsorption performance is considered acceptable. Conversely, if the cells adhere to the wall and spread out or loosely aggregate, the adsorption performance is considered poor. The culture container of this application includes a mounting box and a sealing cover. Several culture chambers are uniformly arranged along the length of the mounting box, and several microcavities are provided at the bottom of each culture chamber. Cells naturally settle and aggregate in the microcavities to form spheres. The synergistic effect of the triple quality control system and the microcavity structure of the container ensures that each batch of products meets the preset standards in the three core indicators of surface smoothness, light transmittance, and ultra-low adsorption performance of cells. This provides a full-process quality assurance for the uniformity of 3D cell sphere formation, high-definition imaging, and high reproducibility of experimental data.

[0033] Furthermore, to further improve cell aggregation and observability within the culture vessel, it is necessary to ensure the vessel surface is smooth after coating with the coating solution. This prevents the formation of pits, cracks, or raised particles that would hinder cell aggregation and spherical formation, thus affecting observation results. Therefore, after coating, the smoothness of the culture vessel surface must be guaranteed. Surface protrusions are detected using a projection light path along the surface to be tested. This projection light path is generated through a light exit port, and the trajectory of the exit port matches the cross-sectional trajectory of the surface to be tested. The projection light path moves towards the surface to be tested. If a protrusion exists on the surface, it will be detected as the projection light path moves... During the process, the projection light path corresponding to the protrusion position will be blocked in advance and detected by the signal receiving port, thereby determining that there is a protruding particle at the current position. Conversely, if all the signal receiving ports lose signals at the same time, it is determined that the light output port is blocked by the culture container, and there is no protrusion on the surface of the culture container. Similarly, by setting a light output port on the surface to be tested of the culture container, the mesh detection light is projected perpendicularly to the surface to be tested of the culture container. If there are depressions or cracks on the surface, the light projected at the cracks will be distorted and deflected. By setting an image acquisition device to collect and detect the mesh detection light, it is possible to conveniently obtain the flatness data of the culture container surface and prevent the depressions and protrusions on the culture container surface from affecting the cell culture effect.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any ordinary changes and substitutions made by those skilled in the art within the scope of the technical solution of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for fabricating a 3D cell spheroid forming container, characterized in that, Includes the following steps: S1. Container substrate cleaning treatment: Select conventional polystyrene culture plates, petri dishes, and matrix plates as substrates, and use sterile deionized water and ethanol solution for ultrasonic cleaning to remove residual impurities on the substrate surface. S2. Container substrate drying treatment: The cleaned container substrate is placed in a sterile and dust-free oven for low-temperature drying, and the substrate surface is activated by low-temperature plasma to form a culture container. After the treatment, it is necessary to ensure the flatness of the substrate and that there are no scratches or protrusions on the surface. S3. Precise preparation of phosphoric acid choline coating solution: Biocompatible phosphoric acid choline polymers are selected as the core functional raw materials, and combined with anhydrous ethanol and sterile buffer solution to prepare a homogeneous and transparent functional coating solution. S4. High-precision flat coating: Through a precision dip coating process, the surface of the pretreated culture container is fully and uniformly coated to form a liquid film of uniform thickness on the surface of the culture container. S5. Drying and curing molding process: The coated culture container is placed in a dust-free environment simulation device with constant temperature and humidity. First, the temperature in the device is adjusted to 37℃, and the liquid film is leveled and shaped by a slow shaking process on an ultra-low speed platform. After the shaping is completed, the temperature is gradually increased to 50℃-70℃ within a period of 1-2 hours to achieve full curing of polymer molecules on the surface of the culture container. S6. Post-processing and quality control testing: After curing, the culture container is sterilized with ethylene oxide. At the same time, the surface flatness, light transmittance, and cell ultra-low adsorption of the culture container are tested. If the culture container passes the test, it is shipped and stored. Otherwise, the culture container is reworked based on the unqualified performance.

2. The method for fabricating a 3D cell spheroid forming container according to claim 1, characterized in that, Step S3 also includes a method for preparing the coating liquid. The steps include: S31, dissolving the biocompatible phosphoric acid choline polymer in anhydrous ethanol to form a stock solution of 8 mg / mL; S32. Allow the stock solution to stand so that the biocompatible phosphoric acid choline polymers are completely dissolved in anhydrous ethanol. S33. After dissolution, dilute the stock solution to 1.6 mg / mL with sterile buffer and anhydrous ethanol, with the volume ratio of anhydrous ethanol to buffer being 1:

1. S34. Allow the diluted stock solution to stand to ensure that the added anhydrous ethanol and buffer solution are fully dissolved.

3. The method for fabricating a 3D cell spheroid forming container according to claim 1, characterized in that, Step S4 further includes the following steps: S41, immersing the processed culture container into the prepared stock solution at a uniform speed without vibration; S42. After the culture container is immersed, keep it still in the stock solution to allow the coating solution to fully wet the substrate surface; S43. Based on the required coating thickness, pull it out of the reservoir at a constant speed of 1cm / min-30cm / min without vibration.

4. The method for fabricating a 3D cell spheroid forming container according to claim 1, characterized in that, Step S5 further includes the following steps: S51, In an environment of 37°C, the coated culture container is placed on a constant temperature and humidity shaker and kept horizontal to ensure that the liquid film on the surface of the culture container is evenly distributed under the action of gravity. S52. Start the shaker to start the shaker at an ultra-low speed and shake slowly, so that the coating liquid flows slowly and continuously on the substrate surface to eliminate local thickness unevenness, and run continuously for 13 hours. S53. After the low-temperature slow shaking process is completed, the processed culture container is transferred to a constant temperature dust-free oven, heated to 50°C at a rate of 1°C / min and maintained for 30 min, then heated to 70°C at a rate of 0.5°C / min and maintained for 60 min. S54. After curing, allow the coating to cool naturally to room temperature to prevent stress cracking.

5. A method for fabricating a 3D cell spheroid forming container according to claim 1, characterized in that, Step S6 further includes the following steps: S61, placing the packaged culture containers inside the sterilizer to ensure that ethylene oxide, heat and humidity can fully penetrate all products; S62. A biological indicator is installed inside the sterilization cabinet to detect the sterilization effect; S63. During the sterilization process, various parameters inside the sterilization cabinet are monitored to obtain the sterilization time, pressure, humidity, temperature, and ethylene oxide consumption inside the sterilization cabinet. S64. After sterilization, transfer the processed product to the testing equipment and perform forced ventilation analysis on the testing equipment for a duration of t. S65. After time period t, the residual amount of ethylene oxide in the detection equipment is detected, and the detection value is X. If X > 10 μg / g, it is determined that the current residual amount of ethylene oxide is too high, and ventilation is continued. Otherwise, it is determined that the current residual amount of ethylene oxide in the culture container is low, and the process jumps to S66 to perform physical performance testing. S66. Linearly scan the surface of the culture container using a profilometer to obtain the surface profile of the culture container. At the same time, inspect the surface profile. If no particles or cracks appear on the surface of the culture container, the surface flatness of the current culture container is deemed to be qualified, and proceed to S67 to continue the inspection. Otherwise, re-coat the surface of the culture container. S67. Using uncoated containers of the same material as a blank control group, and setting the transmittance of the control group to 100%, the culture containers after coating are tested for visible light, and the transmittance of the culture containers is judged. If the transmittance of the culture container is ≥95% relative to the transmittance of the control group, the transmittance of the current culture container is judged to be qualified, and the test is skipped to S68. Otherwise, the surface of the culture container is recoated. S68. The cells to be cultured are seeded at an appropriate density inside a coated culture container, cultured with suitable environmental parameters, and the morphology of the cells is detected. If the cells are suspended in a tightly packed spherical aggregate in the culture container, the current culture container is judged to have good adsorption. Conversely, if the giant cells are attached to the wall and spread out or loosely aggregated, the current culture container is judged to have poor adsorption.

6. A method for fabricating a 3D cell spheroid forming container according to claim 5, characterized in that, Step S66 further includes surface contour flatness detection, including a protruding particle detection step: S661, On the surface of the culture container, a plurality of projection light paths are provided parallel to the surface of the culture container, one end of the projection light path is a light outlet, and the other end is a signal receiving port; S662. The projection optical path is suspended above the surface of the culture container to be tested and moves along the direction of the culture container to be tested. During the movement, the signal receiving port receives the optical signal. S663. If all of the signal receiving ports lose light signals at the same time, it is determined that no protruding particles appear on the surface of the current culture container. Conversely, if one or more of the signal receiving ports lose light signals, it is determined that there are protrusions on the surface of the culture container corresponding to the projection light path. And the crack detection steps: S664, set the light outlet at a position perpendicular to the surface of the culture container to be tested, with the light outlet facing the direction of the culture container and projecting a mesh detection light beam in the direction of the culture container; S665. An image acquisition device is installed on the light outlet. During the projection of light, the image acquisition device acquires the light information on the surface of the culture container. S666. If the light on the surface of the culture container is distorted or deflected, it is determined that the culture container at the current position has a dent or crack; otherwise, it is determined that the surface of the current culture container is flat.

7. A culture container suitable for use with the method for fabricating a 3D cell spheroid formation container as described in any one of claims 1-6, characterized in that, The culture container includes a mounting box and a sealing cover plate disposed on the top of the mounting box. The mounting box is provided with a plurality of culture chambers evenly distributed along its length. Each culture chamber has a plurality of microcavities at its bottom. The cells settle and aggregate within the enclosure to form spheres.

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