Method for manufacturing 3d cell culture plate and 3d cell culture plate
By setting a scaffold within the culture wells of a 3D cell culture plate and using a specific solution to form a flat scaffold, the concave liquid surface problem was solved, achieving uniform cell distribution and clear microscopic imaging, thus improving the stability of high-throughput screening and the cell growth environment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SINOBIOPRINT (SHANGHAI) BIOTECH LTD
- Filing Date
- 2023-12-22
- Publication Date
- 2026-06-19
AI Technical Summary
Existing 3D cell culture plates tend to form concave liquid surfaces after the addition of liquid culture medium, resulting in a reduction in the number of cells in the center of the well, blurred imaging, and poor stability in high-throughput screening.
A scaffold is formed by mixing 10% methacrylamide gelatin with a 1.6% polyethylene oxide solution and placing a scaffold inside the culture well. A limiting ring separates the receiving cavity from the well wall, and the top of the scaffold is flush with the well wall. After the solution is cured by light, a flat scaffold is formed. The scaffold is fixedly connected to the culture well to avoid a concave liquid surface.
It achieves uniform cell distribution in the central region of the scaffold, clear microscopic imaging, improves the stability and imaging quality of high-throughput screening, promotes nutrient exchange, and is suitable for high-throughput screening.
Smart Images
Figure CN117734075B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell, tissue and organoid culture technology, and particularly to a method for manufacturing a 3D cell culture plate and the 3D cell culture plate. Background Technology
[0002] In existing in vitro cell, tissue, and organoid culture techniques, culture methods are divided into suspension culture and adherent culture, the latter further divided into 2D (planar) and 3D (stereoscopic) culture. 2D adherent culture is a commonly used technique for cell expansion; however, cells in 2D culture cannot easily simulate the in vivo 3D environment. Cell-matrix interactions are limited, and cells are subject to surface tension, which can lead to cell differentiation. To overcome these drawbacks, researchers have begun to employ 3D culture methods that more closely resemble the in vivo environment. Unlike traditional 2D cell culture, 3D cell culture recreates the in vivo cellular environment. The multi-layered three-dimensional structure can create gradients of oxygen, nutrients, metabolites, and soluble signals, thereby forming diverse cell populations, tissues, or organs.
[0003] Currently, two methods are commonly used in 3D cell culture technology: scaffold-free and scaffold-based culture. Scaffold-free 3D culture primarily involves allowing cells to self-aggregate into spheroids. However, these self-aggregated cell spheroids vary in size and spatial distribution, with significant differences between wells, leading to poor stability in high-throughput screening. Scaffold-based 3D culture, on the other hand, effectively simulates cell-cell interactions and cell-extracellular matrix interactions in the in vivo environment, while allowing cells to aggregate, proliferate, differentiate, and migrate on the scaffold.
[0004] However, regardless of whether a 3D culture plate contains a scaffold or not, after adding liquid culture medium, capillary action—the adhesion of liquid to the well wall surface—causes the liquid near the wall to rise, forming a concave meniscus. The angle between the liquid surface further away from the well wall and the well wall is less than 90 degrees. This results in more cells and culture medium adhering to the well wall, leading to a significant reduction in cell number in the well center and cell aggregation at the well edges. Furthermore, due to the uneven liquid surface, microscopic imaging is blurred, especially in 96-well and 384-well plates used for high-throughput screening. The smaller the well, the more pronounced the concave meniscus and the more blurred the imaging.
[0005] To address the issue of concave menisci, existing technologies have employed methods such as coating the well walls to alter the contact angle and reduce concave menisci formation, or adding a well plate design to the surface coating. This involves coating the upper half of the well with a hydrophobic layer and designing the upper half of the well wall with a serrated shape to further reduce capillary-induced concave menisci. However, without a scaffold within the well, cells aggregate spontaneously, resulting in cell spheroids of varying sizes, uneven spatial distribution, and significant differences between wells. Furthermore, the lack of a scaffold makes it difficult to provide 3D support for the cells. When a scaffold is used within the well for 3D cell support, the shape of the scaffold itself is difficult to control, leading to inconsistent cell positions within the scaffold. This is detrimental to microscopic observation and, when a fixation device is used to fix the scaffold in the confined space of the culture well, a concave menisci can form at the connection point, causing cells to aggregate towards the fixation device and hindering imaging, thus compromising usability. Summary of the Invention
[0006] The technical problem to be solved by the present invention is to overcome the defect that the culture wells in the cell culture plates of the prior art have concave liquid surfaces that are not conducive to cell culture, and to provide a method for manufacturing a 3D cell culture plate and a 3D cell culture plate.
[0007] The present invention solves the above-mentioned technical problems through the following technical solution:
[0008] A method for manufacturing a 3D cell culture plate, the method being used to manufacture cell culture plates, specifically including the following steps:
[0009] S1. Prepare 10% methacrylamide gelatin and 1.6% polyethylene oxide solution for scaffold preparation, mix the solutions at a 1:1 ratio, and vortex for 5-20 seconds.
[0010] S2. Take a cell culture plate, place the limiting ring of the casting mold into the culture well of the cell culture plate, fit the limiting ring against the bottom of the culture well, and fit the outer periphery of the limiting ring against the wall of the culture well, set the top of the limiting ring flush with the inner wall of the receiving cavity where the limiting ring is used to receive the scaffold, and then pour the vortexed solution into the receiving cavity according to the preset value that the top of the limiting ring is flush with the height of the limiting ring, and oscillate to make the solution spread evenly;
[0011] S3. Irradiate the solution in the containment cavity with light to form a scaffold and solidify it. Clean the solidified scaffold with ultrasonic cleaning and remove the limiting ring. Then, place the cell culture plate with the scaffold into a freeze dryer for freeze drying and sterilize the cell culture plate after freeze drying.
[0012] In this scheme, a solution is prepared so that the scaffold can form pores for cell culture inside itself after casting. The solution is thoroughly mixed and vortexed to make the pores in the scaffold more evenly distributed after casting. Before casting the scaffold, a limiting ring is placed into the culture well of the cell culture plate to isolate the receiving cavity from the well wall. The top of the limiting ring is flush with the inner wall of the receiving cavity. Then, the solution is poured into the receiving cavity for scaffold casting. It should be noted that the sides of the scaffold are flush with the inner wall of the receiving cavity, and the top of the scaffold is flush with the top of the limiting ring by the amount of solution injected into the receiving cavity. To make the scaffold suitable for high-throughput screening cell culture, the amount of solution is cast according to the preset value, which can prevent the top of the scaffold from being sunken or bulging due to inaccurate casting volume. During scaffold casting, the solution can be spread evenly by agitation. After illumination, the solution solidifies to form the scaffold. By removing the retaining ring, the cell culture plate is finally formed with the scaffold. The scaffold ensures 3D cell culture. The top of the scaffold is flush, allowing cells to accumulate mainly in the central area. The scaffold is directly formed in the central area of the bottom of the culture well via the retaining ring. This allows for focused observation of the central area of the scaffold when observing expanded cells, enabling the microscope to observe cell culture through the flush scaffold. The imaging is clear and the position is fixed. Compared to the concave liquid surface, the imaging quality is improved, and a rich sample is provided for high-throughput screening, preventing cells in multiple culture wells from being unobservable and affecting the stability of high-throughput screening.
[0013] Furthermore, the scaffold is directly solidified in the central area at the bottom of the culture plate, fitting snugly and adhering well to the bottom. This facilitates transportation while preventing the scaffold from shifting position within the culture wells. A gap forms between the side of the scaffold and the well wall after the retaining ring is removed, which promotes the exchange of nutrients and metabolites, thus fostering cell growth.
[0014] Preferably, step S3 further includes the following steps:
[0015] S31. After the bracket is shaped and cured, the top and sides of the bracket are drilled and the surface is cut. After drilling and cutting, the bracket is ultrasonically cleaned.
[0016] In this solution, the holes in the top or side of the scaffold are cleared by inserting holes to increase the speed at which cells enter the scaffold during culture. After insertion, the surface is cut to keep the top and sides of the scaffold flush. Residual debris is removed by ultrasonic cleaning to avoid damaging the scaffold.
[0017] Preferably, step S1 further includes the following steps:
[0018] S11. When preparing the solution, the diameter of the pores in the scaffold is kept within the range of 50 to 400 µm by adjusting the freezing rate of the hydrogel precursor solution after mixing the methacrylamide gelatin and the polyethylene oxide.
[0019] In this scheme, the diameter of the pores is changed by adjusting the freezing rate of the precursor solution, so that the scaffold can still be used for culturing different cells, tissues, or organoids, thus improving the applicability of the cell culture plate.
[0020] A 3D cell culture plate is manufactured using the aforementioned method. The 3D cell culture plate has multiple culture wells arranged in an array. It can be used for high-throughput cell screening. A scaffold is disposed within each culture well. The scaffold is cast into the central region of the bottom of each culture well and forms an integral part with it. The casting process creates pores for cell culture within the scaffold. These pores are arranged horizontally and vertically within the scaffold. The top and sides of the scaffold are flush, and the sides of the scaffold form a gap with the pore walls. The top of the scaffold is configured such that when cells and culture medium are injected into the scaffold, the cells and culture medium are flush and evenly distributed at various positions on the top of the scaffold.
[0021] In this method, a scaffold is placed inside the culture wells of a cell culture plate to facilitate 3D cell culture. The top and sides of the scaffold are flush to ensure that the cell suspension spreads evenly on the top of the scaffold when it comes into contact with it, avoiding the situation where cells are concentrated at the edges of the scaffold while the central area has few cells (a "concave liquid surface"). In addition, a gap is formed between the scaffold and the well wall, which promotes the exchange of nutrients and metabolites, thus promoting cell growth. This method is simple to prepare, convenient to operate, and inexpensive, making it particularly suitable for observing and analyzing cell samples from multiple wells during high-throughput screening. Cells in each well can grow effectively, and compared to other cell culture plates with or without scaffolds, cells can be observed more clearly under a microscope, ensuring stability under high-throughput screening.
[0022] Preferably, the 3D cell culture plate further includes a casting mold, which is disposed in the gap and removed from the culture well after the scaffold is formed. The bottom of the casting mold is configured such that when the scaffold is cast, the bottom of the casting mold maintains a closed connection between the scaffold and the wall of the culture well.
[0023] In this method, a casting mold is used to cast the scaffold, fixing it securely to the cell culture plate and preventing it from floating in the culture wells. The casting mold also ensures that the sides of the scaffold are flush with the well walls, further avoiding the "concave liquid surface" phenomenon. After casting, the casting mold is promptly removed to prevent interference with nutrient exchange during cell culture.
[0024] Preferably, the casting mold includes a limiting ring with a receiving cavity. The top of the limiting ring is flush with the sidewalls of the receiving cavity. When the support is cast into the receiving cavity, the amount of solution cast into the support is based on a preset value that is flush with the top height of the limiting ring.
[0025] In this design, a limiting ring is positioned in the gap between the bracket and the hole wall. The limiting ring has a receiving cavity, allowing the bracket to be poured into the receiving cavity and spaced apart from the hole wall. By setting the top of the limiting ring flush with the side wall of the receiving cavity, the top and side of the bracket after casting are flush. It should be noted that, to ensure the top of the bracket is flush, the amount of solution used for pouring the bracket during casting must be according to the preset amount that is flush with the top of the limiting ring, in order to avoid unevenness on the surface of the bracket.
[0026] Preferably, the casting mold further includes a support strip and a connecting arm. The support strip is disposed on the top of the bottom plate of the 3D cell culture plate and covers the culture hole. The support strip is used to engage with the top of the bottom plate of the 3D cell culture plate. One end of the connecting arm is connected to the top of the limiting ring and disposed away from the receiving cavity. The other end of the connecting arm is connected to the support strip.
[0027] In this design, the casting mold can also be equipped with support bars and connecting arms. The support bars, connecting arms, and limiting rings are connected in sequence. The supporting bars drive the connecting arms to remove the limiting rings from the culture wells, so that a cell culture plate with only a scaffold is formed after casting. By setting up support bars and connecting arms, the situation where a limiting ring alone might get stuck in the culture well when it is removed is avoided.
[0028] Preferably, the limiting ring is a permanent magnet.
[0029] In this solution, the limiting ring is set as a permanent magnet, so that it can be removed from the culture well in a timely manner through electromagnetic adsorption or reverse repulsion, avoiding the situation where the limiting ring may get stuck in the culture well when it is removed.
[0030] Preferably, the diameter of the hole is in the range of 50 to 400 µm.
[0031] In this approach, the diameter of the pores is limited to meet the needs of cell attachment and migration. The diameter of the pores is not less than 50 µm to avoid the pores being too small and blocking the cells from passing through, thus ensuring the cell culture efficiency.
[0032] Preferably, the scaffold is made of hydrogel, which comprises a 1:1 mixture of methacrylated gelatin and polyethylene oxide solution, wherein the concentration of the methacrylated gelatin is in the range of 10% to 20%, and the concentration of the polyethylene oxide is in the range of 1% to 2%.
[0033] In this scheme, a hydrogel solution containing methacrylamide gelatin and polyethylene oxide is prepared, wherein polyethylene oxide is a placeholder solution for forming a scaffold, so that pores are formed inside the scaffold after casting, reducing the time required for machining to create pores inside the scaffold and improving the manufacturing efficiency of cell culture plates.
[0034] The significant advantages of this invention are as follows: By configuring the solution, the scaffold can form pores for cell culture within itself after casting. Thorough mixing and vortexing of the solution result in a more uniform distribution of pores within the scaffold after casting. Before casting the scaffold, a limiting ring is inserted into the culture well of the cell culture plate, isolating the receiving cavity from the well wall. The top of the limiting ring and the inner wall of the receiving cavity are also flush. The solution is then poured into the receiving cavity for scaffold casting. It is important to note that the sides of the scaffold are flush with the inner wall of the receiving cavity, and the top of the scaffold is flush with the top of the limiting ring by the amount of solution injected into the receiving cavity. To ensure the scaffold is suitable for high-throughput screening cell culture, the amount of solution is cast according to a preset value, preventing a central depression or convexity at the top of the scaffold. In this case, the solution can be spread evenly by shaking during scaffold casting. After the solution is exposed to light, it solidifies to form a scaffold. By removing the retaining ring, the cell culture plate is finally formed with a scaffold. The scaffold ensures 3D cell culture. At the same time, the top of the scaffold is set to be flush, which allows cells to mainly accumulate in the central area of the scaffold. The scaffold is fixed to the central area of the bottom of the culture well by the retaining ring. Thus, when observing the expanded cells, the central area of the scaffold can be focused on. The microscope can observe the cell culture through the flush scaffold, resulting in clear and fixed images. Compared with the "concave liquid surface" caused by culture without a scaffold, the imaging quality is improved and a rich sample is provided for high-throughput screening. This avoids the inability of cells in multiple culture wells to be observed, which would affect the stability of high-throughput screening.
[0035] Furthermore, the scaffold is directly solidified in the central area at the bottom of the culture plate, fitting snugly and adhering well to the bottom. This facilitates transportation while preventing the scaffold from shifting position within the culture wells. A gap forms between the side of the scaffold and the well wall after the retaining ring is removed, which promotes the exchange of nutrients and metabolites, thus fostering cell growth. Attached Figure Description
[0036] Figure 1 This is a top view of a 3D cell culture plate according to a preferred embodiment of the present invention.
[0037] Figure 2 This is a side view of a 3D cell culture plate according to a preferred embodiment of the present invention.
[0038] Figure 3 This is a diagram showing the positional relationship between the top cover and the bottom plate of a 3D cell culture plate according to a preferred embodiment of the present invention.
[0039] Figure 4 This is a schematic diagram of the structure of a casting mold according to a preferred embodiment of the present invention.
[0040] Figure 5 This is a schematic diagram of the structure of the receiving cavity in a preferred embodiment of the present invention.
[0041] Figure 6 This is a side view of a casting mold according to a preferred embodiment of the present invention.
[0042] Figure 7 This is a schematic diagram of the hole structure of a preferred embodiment of the present invention.
[0043] Figure 8 This is a flowchart illustrating a preferred embodiment of the manufacturing method of a 3D cell culture plate according to the present invention.
[0044] Explanation of reference numerals in the attached figures:
[0045] Cell culture plate 1
[0046] Culture well 11
[0047] Base plate 12
[0048] Bracket 2
[0049] Hole 21
[0050] Casting mold 3
[0051] Limiting ring 31
[0052] Support bar 32
[0053] Connecting arm 33
[0054] Receiving cavity 311
[0055] Top cover 4 Detailed Implementation
[0056] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.
[0057] Example 1
[0058] This embodiment provides a method for manufacturing a 3D cell culture plate, such as... Figure 8 As shown, the manufacturing method of the 3D cell culture plate is used to manufacture cell culture plate 1. The manufacturing method of the 3D cell culture plate specifically includes the following steps:
[0059] S1. Prepare solutions of 10% methacrylamide gelatin and 1.6% polyethylene oxide for the preparation of scaffold 2. Mix the solutions at a ratio of 1:1 and vortex for 5-20 seconds.
[0060] S2. Take cell culture plate 1, place the limiting ring 31 of the casting mold 3 into the culture well 11 of cell culture plate 1, fit the limiting ring 31 with the bottom of culture well 11, and fit the outer periphery of the limiting ring 31 with the wall of culture well 11, set the top of the limiting ring 31 flush with the inner wall of the receiving cavity 311 used to receive the scaffold 2, and then pour the vortexed solution into the receiving cavity 311 according to the preset value that the top height of the limiting ring 31 is flush with the limiting ring 31, and oscillate to make the solution spread evenly.
[0061] S3. Irradiate the solution in the receiving cavity 311 with light to form a scaffold and solidify it. Clean the solidified scaffold 2 with ultrasonic cleaning and remove the limiting ring 31. Then, place the cell culture plate 1 with the scaffold 2 into a freeze dryer for freeze drying and sterilize the cell culture plate 1 after freeze drying.
[0062] Specifically, the solution used for casting the scaffold 2 is prepared using non-cytotoxic methacrylamide gelatin and polyethylene oxide. This ensures that the scaffold 2 can be used for cell culture after molding without affecting cell growth. By limiting the concentration of the methacrylamide gelatin and polyethylene oxide solution, pores 21 can be formed inside the cast scaffold 2. The polyethylene oxide, acting as a site-filling solution for the scaffold 2, also known as a pore-forming agent, dissolves after the scaffold 2 is molded, thus forming pores 21 within the scaffold 2 itself after casting. Figure 7As shown, the pores 21 are irregular, near-circular pores arranged in both horizontal and vertical directions. Thorough mixing and vortexing of the solution, after casting, makes the pores 21 in the scaffold 2 more evenly distributed. The pores 21 allow the cell fluid to reproduce the in vivo environment of the cell when injected into the scaffold 2. The three-dimensional structure of the scaffold 2 can form gradients of oxygen, nutrients, metabolites, and soluble signals, thereby forming diverse cell populations, tissues, or organs. The solution formulation of the scaffold 2 can also use materials already used in the technology for manufacturing the scaffold 2, such as polymer materials (e.g., PLA, PS) and biological materials (e.g., agarose, collagen, fibronectin, gelatin, laminin, etc.), which will not be elaborated further here.
[0063] Before casting the scaffold 2, the limiting ring 31 needs to be placed into the culture wells 11 of the cell culture plate 1. The culture wells 11 are set on the bottom plate 12 of the cell culture plate 1. When making 3D cell culture plates, 24-well, 48-well, and 96-well cell culture plates 1, as well as 8-well or 12-well cells culture plates 1 inserted in rows, can be used. The number and form of the culture wells 11 are existing technologies and will not be described in detail here. The limiting ring 31 used for limiting can be made of stainless steel, materials that can be used to manufacture 3D printing materials, such as PLA or ceramic materials, permanent magnets, and silicone, etc., and the above materials must be treated to be non-cytotoxic. The size of the limiting ring 31 needs to be processed according to the size of the culture wells 11. During processing, 3D printing or mold casting can be used to precisely control the size, so that the top of the limiting ring 31 is flush with the inner wall of the receiving cavity 311. It should be noted that flush means flush in the horizontal direction. When the bottom of the 11 contacts the bottom of the limiting ring 31, it does not affect the fact that the top of the limiting ring 31 and the inner wall of the receiving cavity 311 remain flush. The limiting ring 31 can be used for automated mechanical operation or manual operation when placing and removing the culture well 11 after processing. The limiting ring 31 isolates the communication between the receiving cavity 311 and the wall of the culture well 11, preventing the cast support 2 from connecting with the well wall. Then, the solution is poured into the receiving cavity 311 for casting the support 2. It can be understood that the side of the support 2 passes through the inner wall of the receiving cavity 311... To ensure flush alignment, the top of the scaffold 2 is flush with the top of the limiting ring 31 by injecting solution into the receiving cavity 311. For cell culture plates 1 suitable for high-throughput screening, such as 96-well and 384-well plates, the solution volume is injected according to a preset value to ensure that the scaffold 2 is suitable for high-throughput screening cell culture, avoiding a central depression or bulge at the top of the scaffold 2. This embodiment uses a volume of 30µl for the scaffold 2 as an example, but it is not a limitation. The limiting ring 31 has a cylindrical structure and The central area is provided with a receiving cavity 311, which is also a cylindrical structure, so that the scaffold 2 is cylindrical after casting. The receiving cavity 311 runs through the upper and lower ends of the limiting ring 31. The height of the top of the limiting ring 31 can be pre-processed according to the cells to be cultured, and the volume of the receiving cavity 311 is consistent with the volume of the scaffold 2, so that the liquid level of the solution is flush with the top of the limiting ring 31 after the solution is injected into the receiving cavity 311. A high-precision pipette can be used when injecting the solution, which will not be described in detail here.During the casting of the scaffold 2, the solution can be spread evenly by agitation. After the solution is irradiated with 405 nm light, it solidifies to form the scaffold 2. By removing the limiting ring 31, the cell culture plate 1 is finally formed with the scaffold 2. The scaffold 2 ensures 3D cell culture. At the same time, the top of the scaffold 2 is set to be flush, which allows the cells to mainly accumulate in the central area of the scaffold 2. The scaffold 2 is fixed to the central area of the bottom of the culture well 11 by casting. Thus, when observing the expanded cells, the central area of the scaffold 2 can be focused, allowing the microscope to observe the cell culture through the flush scaffold 2. The imaging is clear and the position is fixed. Compared with the case of a concave liquid surface, the imaging quality is improved and a rich sample is provided for high-throughput screening. It avoids the inability of cells in multiple culture wells to be observed, which affects the stability of high-throughput screening. Furthermore, the 3D cell culture plate manufactured using this method has a shorter screening cycle during cell culture, is easier to achieve high-throughput and automation, and is a clinically relevant drug screening model.
[0064] Furthermore, after casting, the scaffold 2 is fixedly connected to the cell culture plate 1, facilitating transportation while preventing changes in the cell position within the culture wells 11. A gap is formed between the side of the scaffold 2 and the well wall of the culture well 11 after the retaining ring 31 is removed. This gap allows the nutrient solution to be injected into the culture wells 11, promoting the exchange of nutrients and metabolites between the cells in the scaffold 2 and the cells, thus promoting cell growth. Additionally, in this embodiment, the scaffold 2 is coated with various extracellular matrix components, such as collagen, laminin, fibronectin, and matrix gel, which better simulates the in vivo environment and improves the adhesion and differentiation of normal and transformed adherent epithelial cells and other cell types.
[0065] After the scaffold 2 is cast, the interior of the scaffold 2 is cleaned by ultrasonic cleaning. Then, the cell culture plate 1 with the scaffold 2 is placed in a freeze dryer for freeze drying for 12 hours to further improve the stability of the scaffold 2. After freeze drying, the cell culture plate 1 is sterilized.
[0066] Furthermore, step S3 specifically includes the following steps:
[0067] S31. After the bracket 2 is shaped and cured, insert holes and cut the surface of the top and sides of the bracket 2. After inserting holes and cutting the surface, the bracket 2 is then ultrasonically cleaned.
[0068] Specifically, perforations are made in the scaffold 2 using a plum blossom-shaped tool to clear the holes 21 in the flush portion of the top or side of the scaffold 2, increasing the speed at which cells enter the scaffold 2 during culture. After perforation, the surface is trimmed to ensure that the top and sides of the scaffold 2 remain flush, preventing a "concave liquid surface" during cell fluid injection. Ultrasonic cleaning for 30 minutes provides preliminary sterilization and impurity removal from the interior of the scaffold 2 while avoiding damage. Preferably, a parallel laser can be used to detect any unevenness on the surface of the scaffold 2 to ensure that its top and sides remain flush after casting.
[0069] In other embodiments, an excess sample is added to the limiting ring 31 to make the surface bulge. After photocuring, the upper surface is flattened using a planing technique to obtain a completely transparent and flat support 2, so that the microscope image is clearer.
[0070] In this embodiment, step S1 further includes the following steps:
[0071] S11. When preparing the solution, the diameter of the pores 21 of the scaffold 2 is kept within the range of 50~400 µm by adjusting the freezing rate of the hydrogel precursor solution after mixing methacrylamide gelatin and polyethylene oxide.
[0072] Specifically, the freezing rate of the precursor solution can be adjusted using existing adjustment methods and structures, which will not be elaborated on here. By adjusting the freezing rate of the precursor solution, the diameter of the pores 21 can be changed, so that the scaffold 2 can still be used for culturing different cells, thereby improving the applicability of the cell culture plate 1.
[0073] In other embodiments, the pore size 21 can be adjusted by adding ingredients from the prior art that can affect the pore size 21 of the scaffold 2 to the solution in which the scaffold 2 is cast, or by adjusting the mixing ratio of methacrylamide gelatin and polyethylene oxide. This is prior art. It should be noted that the pore size 21 must always be maintained greater than 50µm to avoid affecting cell passage. The above-mentioned ingredients are prior art and will not be elaborated further here. It is understood that, for different cell cultures, photosensitive materials, such as CSMA, HAMA, etc., can also be added to the above solution to suit photosensitive cell growth.
[0074] like Figures 1-7 As shown, this embodiment also provides a 3D cell culture plate, which is manufactured using the above-described method. The 3D cell culture plate has multiple culture wells 11 arranged in an array. The 3D cell culture plate is used for high-throughput cell screening, such as... Figure 2As shown, a support 2 is provided in the culture well 11. The support 2 is poured into the central area of the bottom of the culture well 11 and forms an integral part with the culture well 11. After pouring, the support 2 forms a hole 21 for cell culture. The holes 21 are arranged in the horizontal and vertical directions and are set in the support 2. The top and sides of the support 2 are flush, and the sides of the support 2 form a gap with the pore wall of the culture well 11. The top of the support 2 is set so that when cells and culture medium are injected into the support 2, the cells and culture medium are flush with each other and evenly distributed at various positions on the top of the support 2.
[0075] Specifically, such as Figure 3 As shown, the cell culture plate 1 includes a base plate 12 and a top cover 4. Multiple culture wells 11 are arrayed on the base plate 12, and a locking part is provided on the edge of the base plate 12 near the opening of the culture well 11. The locking part is used to cooperate with the top cover 4 and prevent external dust from entering the culture well 11 during storage or transportation of the cell culture plate 1. A scaffold 2 is poured into the culture well 11 and is fixedly connected to the culture well 11, i.e., fixedly connected to the base plate 12. The pores 21 formed by the scaffold 2 itself allow for cell culture in both horizontal and vertical directions. Compared to 2D cell culture, it can create gradients of oxygen, nutrients, metabolites, and soluble signals, thereby forming diverse cell populations, tissues, or organs, overcoming many shortcomings of single-layer 2D culture. Furthermore, the top and sides of the scaffold 2 are flush. In this embodiment, the scaffold 2 has a cylindrical structure. Of course, in other embodiments, the receiving cavity 311 can also be a cuboid structure or other shapes with flush surfaces, which will not be elaborated further here. The flush top and sides allow cells and nutrient solution to spread evenly on the top of the scaffold 2 upon contact, preventing cells from concentrating at the edges while the central area remains sparsely populated (a "concave liquid surface"). Furthermore, the scaffold 2 forms a gap with the walls of the culture wells 11, facilitating the exchange of nutrients and metabolites and promoting cell growth. This also reduces the manufacturing cost of the cell culture plate 1, simplifying its structure and making it easier to operate and observe during high-throughput screening. Especially during high-throughput screening, it allows for observation and analysis of cell samples from multiple wells 11, ensuring effective cell growth within each well. Compared to other cell culture plates 1 with or without scaffolds, cell growth is more consistent with its original environment, resulting in more uniform cell colonies. Clearer cell observation under a microscope ensures stability during high-throughput screening.
[0076] In other embodiments, the hydrophobic scaffold 2 is subjected to surface modification techniques to enhance cell adhesion. Preferably, the surface modification techniques are physicochemical methods, including plasma treatment and glow discharge treatment, to further improve cell adhesion.
[0077] In this embodiment, the 3D cell culture plate also includes a casting mold 3, which is disposed in the gap and removed from the culture hole 11 after the scaffold 2 is formed. The bottom of the casting mold 3 is configured such that when the scaffold 2 is cast, the bottom of the casting mold 3 maintains the communication between the scaffold 2 and the hole wall of the culture hole 11.
[0078] Specifically, the casting mold 3 is used to cast the scaffold 2 into the culture wells 11. After casting, the casting mold 3 is removed promptly to avoid affecting the exchange of nutrients during cell culture. This ensures that the manufactured cell culture plate 1, in appearance, only includes the scaffold 2, the base plate 12, and the top cover 4. Since the cells and culture medium can remain flush with the scaffold 2 instead of accumulating at its edges when injected, the outer periphery of the scaffold 2 must be isolated from the well walls during casting to prevent liquid from accumulating on the well walls due to surface tension, thus avoiding the problem of a "concave liquid surface." A "concave liquid surface" not only affects cell growth but also affects microscope imaging when observing cells. By casting the scaffold 2 using the casting mold 3, the scaffold 2 is fixedly connected to the cell culture plate 1, preventing the scaffold 2 from suspending in the culture wells 11 and reducing the difficulty of microscope imaging.
[0079] Furthermore, in this embodiment, the casting mold 3 includes a limiting ring 31, the limiting ring 31 has a receiving cavity 311, the top of the limiting ring 31 is flush with the side wall of the receiving cavity 311, and when the support 2 is poured into the receiving cavity 311, the amount of solution for pouring the support 2 is poured into the receiving cavity 311 according to a preset value that is flush with the top height of the limiting ring 31.
[0080] Specifically, the limiting ring 31 is disposed in the gap between the scaffold 2 and the hole wall. The limiting ring 31 has a receiving cavity 311, so that the scaffold 2 can be poured into the receiving cavity 311 and spaced apart from the hole wall. The top of the limiting ring 31 is flush with the top of the scaffold 2, so that the height of the scaffold 2 can be adjusted by the limiting ring 31. By making the side wall of the receiving cavity 311 flush, the top and side of the cast scaffold 2 are flush. It should be noted that in order to ensure that the top of the scaffold 2 is flush, the amount of solution used to pour the scaffold 2 during pouring should be according to the preset amount that is flush with the top of the limiting ring 31, so as to ensure that the top of the scaffold 2 will not be uneven when cells and culture medium are injected.
[0081] In other embodiments, such as Figure 4 , Figure 5 and Figure 6As shown, the casting mold 3 also includes a support strip 32 and a connecting arm 33. The support strip 32 is placed on the top of the bottom plate 12 of the 3D cell culture plate and covers the culture hole 11. The support strip 32 is used to engage with the top of the bottom plate 12 of the 3D cell culture plate. One end of the connecting arm 33 is connected to the top of the limiting ring 31 and is located away from the receiving cavity 311. The other end of the connecting arm 33 is connected to the support strip 32.
[0082] Specifically, the support bar 32, the connecting arm 33, and the limiting ring 31 are fixedly connected in sequence. The support bar 32 is located above the connecting arm 33 and the limiting ring 31. The support bar 32 is a flat plate and has a corresponding flange for the engaging part of the base plate 12 to engage the support bar 32 on the base plate 12. The connecting arm 33 is a cylindrical structure and is located between the support bar 32 and the limiting ring 31. The connection between the connecting arm 33 and the limiting ring 31 is located away from the receiving cavity 311 to avoid the solution part of the support 2 sticking together during the casting of the support 2 and affecting the flatness of its top. During use, the support bar 32 is engaged with the top of the base plate 12 and covers the culture well 11. The connecting arm 33 and the limiting ring 31 extend into the culture well 11 respectively. Solution is injected into the limiting ring 31 to pour the scaffold 2. After pouring, the limiting ring 31 is removed from the culture well 11 by the connecting arm 33 driven by the support bar 32, so that a cell culture plate 1 with only the scaffold 2 is formed after pouring. By setting the support bar 32 and the connecting arm 33, the situation that the limiting ring 31 may get stuck in the culture well 11 when it is removed is avoided.
[0083] In other embodiments, the limiting ring 31 is a permanent magnet.
[0084] Specifically, by setting the limiting ring 31 as a permanent magnet, it can be removed from the culture well 11 in a timely manner through electromagnetic adsorption or reverse repulsion. For example, after the limiting ring 31 is placed in the culture well 11, it is placed at the bottom of the culture well 11 by electromagnetic adsorption. After the pouring is completed, the power is turned off and the cell culture plate 1 is inverted to remove the limiting ring. Alternatively, a magnetic rod corresponding to the culture well 11 can be used to insert into the culture well 11 and pull out the limiting ring 31. This avoids the situation where the limiting ring 31 may get stuck in the culture well 11 when it is removed when it is set alone. Compared with the method of setting support bar 32 and connecting arm 33, it is lower in cost and simpler in structure.
[0085] In another embodiment, the limiting ring 31 can also be made of a metal material that can be attracted by an electromagnet. In this case, the limiting ring 31 is placed into the culture well 11 and the electromagnet is used to hold the limiting ring 31 while the scaffold 2 is poured. After pouring, the cell culture plate 1 is inverted to remove the limiting ring 31. The purpose of this is to remove the limiting ring 31 in time after the scaffold 2 is poured. This will not be elaborated further here.
[0086] In this embodiment, as Figure 7As shown, the diameter of hole 21 ranges from 50 to 400 µm.
[0087] Specifically, the pores 21 in the scaffold 2 are formed directly after casting, avoiding the problem of excessively long manufacturing cycles for the cell culture plate 1 caused by the need for additional processing of the pores 21 in the scaffold 2. By limiting the diameter of the pores 21 to meet the needs of cell attachment and migration, the diameter of the pores 21 is not less than 50 µm to avoid the situation where the pores 21 are too small and block the passage of cells, thus ensuring cell culture efficiency. Preferably, the diameter of the pores 21 is in the range of 100-300 µm.
[0088] In this embodiment, the scaffold 2 is made of hydrogel, which includes a 1:1 mixture of methacrylamide gelatin and polyethylene oxide solution, wherein the concentration of methacrylamide gelatin ranges from 10% to 20%, and the concentration of polyethylene oxide ranges from 1% to 2%.
[0089] Specifically, by configuring a hydrogel solution containing methacrylamide gelatin and polyethylene oxide, pores 21 are formed inside the scaffold after casting, reducing the time required for machining the pores inside the scaffold 2 and improving the manufacturing efficiency of the cell culture plate 1. Preferably, the concentration of methacrylamide gelatin is 10%, and the concentration of polyethylene oxide is 1.6%.
[0090] Example 2
[0091] This embodiment provides a detection method for removing the influence of "concave liquid surface" in 3D cell culture plates, for testing the 3D cell culture plate in Embodiment 1, specifically including the following steps:
[0092] First, cells were cultured in scaffold 2 and stained.
[0093] Secondly, the stained cells were seeded into this 3D cell culture plate and a regular 3D culture plate containing the influence of the "concave liquid surface".
[0094] Cell colonies were then observed after standing for 24 h and 48 h.
[0095] Finally, the culture plate containing the "concave liquid surface" and the 3D cell culture plate were observed under a microscope.
[0096] The above detection method shows that cells in a culture plate with a concave liquid surface are more distributed at the edge of the culture plate, while fewer cells are in the central area of the culture plate. In contrast, cells cultured using this 3D cell culture plate are evenly distributed on the scaffold 2, and the imaging is clearer.
[0097] Example 3
[0098] This embodiment provides a method for culturing lung cancer cells using a 3D cell culture plate, for testing the 3D cell culture plate in embodiment 1, specifically including the following steps:
[0099] First, A549 cells were cultured on 2D culture plates. After the cells reached confluence, they were digested and counted.
[0100] Secondly, the cell count was 2 × 10⁻⁶. 7 Cells / ml. Centrifuge to precipitate the cells, discard the culture medium, and resuspend the cells to a density of 5 × 10⁶ cells / ml. 6 per ml.
[0101] Next, open the top cover 4, mix the cell suspension, and use a pipette to draw 150 µl of cell suspension and add it dropwise from directly above the support 2.
[0102] Next, after completion, close the top cover 4 and incubate at 37℃ in a 5% CO2 incubator for 1 h.
[0103] Then, preheat the culture medium, add the culture medium along culture well 11 after incubation, and continue culturing.
[0104] Finally, all the above steps were performed under sterile conditions.
[0105] It should be noted that the cell culture plate 1 in this embodiment is a 48-well plate with dimensions of 1 cm long × 1 cm wide × 2 mm thick. Through the above culture method and observation of the cells, uniform cell growth and distribution, clear microscopic observation, and structural stability can be achieved. This better promotes the exchange of nutrients and metabolites, improves cell growth efficiency and stability, and is more conducive to high-throughput screening.
[0106] Example 4
[0107] This embodiment provides a method for culturing mouse small intestinal organoids using a 3D cell culture plate, for testing the 3D cell culture plate in embodiment 1, specifically including the following steps:
[0108] First, the mice were euthanized by cervical dislocation, and a 15cm segment of intestine from the terminal ileum was collected and placed in PBS washing solution at 4°C.
[0109] Next, use pointed forceps to remove the membranes, blood vessels, and fat outside the intestine, and use a syringe to inject PBS into one end of the small intestine for rinsing.
[0110] Subsequently, cells were extracted using existing techniques for extracting digested tissue cells, cell counting was performed, and the cells were then centrifuged and resuspended to achieve a cell density of 5 × 10⁻⁶. 6 per ml.
[0111] Finally, select a suitable-sized 3D cell culture plate, and seed the cells directly into scaffold 2 without using matrix gel to mix them, for subsequent culture and observation. This reduces the cost of culture.
[0112] Example 5
[0113] This embodiment provides a method for culturing tumor cells using a 3D cell culture plate, for testing the 3D cell culture plate in embodiment 1, specifically including the following steps:
[0114] First, the obtained tumor tissue was cleaned of fat, connective tissue, and necrotic parts. It was then washed three times with Hanks' solution in a petri dish, and the tissue was minced and cut into 1-2 mm pieces. 3 Small pieces.
[0115] Next, add 0.25% trypsin or 2000 U / ml collagenase.
[0116] Then, digest in a 37°C water bath for 30 min or longer, remove the digestion solution, wash 3 times with washing solution, wash once with culture medium, resuspend in complete culture medium, and disperse by pipetting to prepare a cell suspension, and count.
[0117] Finally, cells can be directly seeded into scaffold 2 without the need for matrix gel mixing, for subsequent culture and observation, enabling high-throughput drug screening and reducing culture costs. It should be noted that the cell culture plate used in this embodiment is a 96-well plate to ensure high-throughput screening requirements.
[0118] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.
Claims
1. A method for manufacturing a 3D cell culture plate, wherein the method is used to manufacture cell culture plates, characterized in that, The manufacturing method of the 3D cell culture plate specifically includes the following steps: S1. Prepare 10% methacrylamide gelatin and 1.6% polyethylene oxide solution for scaffold preparation, mix the solutions at a 1:1 ratio, and vortex for 5-20 seconds. S2. Take a cell culture plate, place the limiting ring of the casting mold into the culture well of the cell culture plate, fit the limiting ring against the bottom of the culture well, and fit the outer periphery of the limiting ring against the wall of the culture well, set the top of the limiting ring flush with the inner wall of the receiving cavity where the limiting ring is used to receive the scaffold, and then pour the vortexed solution into the receiving cavity according to the preset value that the top of the limiting ring is flush with the height of the limiting ring, and oscillate to make the solution spread evenly; S3. Irradiate the solution in the containment cavity with light to form a scaffold and solidify it. Clean the solidified scaffold with ultrasonic cleaning and remove the limiting ring. Then, place the cell culture plate with the scaffold into a freeze dryer for freeze drying and sterilize the cell culture plate after freeze drying.
2. The method for producing a 3D cell culture plate according to claim 1, wherein Step S3 further includes the following steps: S31. After the bracket is shaped and cured, the top and sides of the bracket are drilled and the surface is cut. After drilling and cutting, the bracket is ultrasonically cleaned.
3. The method for producing a 3D cell culture plate according to claim 1, wherein Step S1 further includes the following steps: S11. When preparing the solution, the diameter of the pores in the scaffold is kept within the range of 50 to 400 µm by adjusting the freezing rate of the hydrogel precursor solution after mixing the methacrylamide gelatin and the polyethylene oxide.
4. A 3D cell culture plate, characterized by, The 3D cell culture plate is manufactured using the method described in any one of claims 1-3. The 3D cell culture plate has multiple culture wells arranged in an array. The 3D cell culture plate is used for high-throughput cell screening. A scaffold is disposed within each culture well. The scaffold is cast into the central region of the bottom of each culture well and forms an integral part with the culture well. The scaffold forms pores for cell culture after casting. The pores are arranged horizontally and vertically within the scaffold. The top and sides of the scaffold are flush, and the sides of the scaffold form a gap with the pore wall of the culture well. The top of the scaffold is configured such that when cells and culture medium are injected into the scaffold, the cells... The cells and the culture medium are flush with each other and evenly distributed at various positions on the top of the scaffold. The 3D cell culture plate also includes a casting mold, which is disposed in the gap and removed from the culture well after the scaffold is formed. The bottom of the casting mold is configured to maintain a closed communication between the scaffold and the pore wall when the scaffold is cast. The casting mold includes a limiting ring with a receiving cavity. The top of the limiting ring is flush with the scaffold, and the side walls of the receiving cavity are flush with the scaffold. When the scaffold is cast into the receiving cavity, the amount of solution cast into the receiving cavity is based on a preset value that is flush with the top height of the limiting ring.
5. The 3D cell culture plate of claim 4, wherein, The casting mold also includes a support strip and a connecting arm. The support strip is placed on top of the 3D cell culture plate and covers the culture hole. The support strip is used to engage with the top of the bottom plate of the 3D cell culture plate. One end of the connecting arm is connected to the top of the limiting ring and is located away from the receiving cavity. The other end of the connecting arm is connected to the support strip.
6. The 3D cell culture plate as described in claim 5, characterized in that, The limiting ring is a permanent magnet.
7. The 3D cell culture plate of claim 4, wherein, The diameter of the hole ranges from 50 to 400 µm.
8. The 3D cell culture plate of claim 4, wherein, The scaffold is made of hydrogel, which comprises a 1:1 mixture of methacrylamide gelatin and polyethylene oxide solution, wherein the concentration of methacrylamide gelatin ranges from 10% to 20%, and the concentration of polyethylene oxide ranges from 1% to 2%.