TWI901194B - Dual-sided multi-cell co-culture system and method of culturing cells - Google Patents

Abstract

A dual-sided multi-cell co-culture system and a method of culturing cells are provided. A dual-sided multi-cell co-culture system includes a first culture tank, a second culture tank, a membrane assembly, a first culture plate and a second culture plate. The membrane assembly is configured to be removably mounted to the first culture tank and configured to be removably mounted to the first culture tank. The first culture plate has at least one well, wherein the at least one well of the first culture plate is configured to receive the first culture tank equipped with the membrane assembly. The second culture plate has at least one well, wherein the at least one well of the second culture plate is configured to receive the second culture tank equipped with the membrane assembly.

Description

Double-sided multi-cell co-culture system and cell culture method

This disclosure relates to a double-sided multi-cell co-culture system and methods for culturing different cell populations.

In the field of cell culture, traditional methods often involve the use of monolayer cell culture systems, which are typically capable of culturing only a single cell type at a time. These systems typically utilize culture dishes or flasks made of materials such as polystyrene to provide a surface for cells to attach and grow. While effective for basic cell culture, these systems face significant limitations when simulating more complex biological environments.

A major challenge of traditional cell culture techniques is the inability to effectively co-culture diverse cell types that require distinct microenvironments. For example, anaerobic bacteria require an oxygen-free environment, while many mammalian cells, including epithelial and endothelial cells, require an oxygen-free environment. Establishing a system capable of simultaneously maintaining these diverse conditions has been a complex and expensive undertaking.

Furthermore, current cell culture systems often require extensive handling and manipulation of the cultures, increasing the risk of contamination and variability in experimental results. This is particularly problematic in applications requiring high precision and reproducibility, such as drug testing and disease modeling.

According to one embodiment of the present disclosure, a double-sided multi-cell co-culture device includes a first culture tank, a second culture tank, and a membrane assembly. The first culture tank includes a generally cylindrical body having a first end and a second end opposite the first end, the first end of the body having a first opening, and the second end of the body having a second opening. The second culture tank includes a generally cylindrical body having a first end and a second end opposite the first end, the first end of the body having a first opening, and the second end of the body having a second opening. The membrane assembly is configured to be removably mounted on the first end of the body of the first culture tank. When the membrane assembly is mounted on the first end of the first culture tank body, the membrane assembly is configured to substantially close the first opening of the first culture tank body. The membrane assembly is configured to be removably mounted on the first end of the second culture tank body. When the membrane assembly is mounted on the first end of the second culture tank body, the membrane assembly is configured to substantially close the first opening of the second culture tank body.

According to another embodiment of the present disclosure, a double-sided multi-cell co-culture system includes a first culture tank, a second culture tank, a membrane assembly, a first culture plate, and a second culture plate. The membrane assembly is configured to be removably mounted on the first culture tank and removably mounted on the second culture plate. The first culture plate has at least one culture well configured to accommodate the first culture tank. When the first culture tank and the membrane assembly are mounted and placed in the at least one culture well of the first culture plate, a first surface of the membrane assembly faces an interior space of the first culture tank, and a second surface of the membrane assembly faces a bottom of the at least one culture well of the first culture plate. The second culture plate has at least one culture well configured to accommodate the second culture tank. When the second culture tank and the membrane assembly are mounted and placed in the at least one culture well of the second culture plate, the second surface of the membrane assembly faces an interior space of the second culture tank, and the first surface of the membrane assembly faces a bottom of the at least one culture well of the second culture plate.

According to another embodiment of the present disclosure, a cell co-culture method includes: installing a membrane assembly in a first culture tank, wherein a first surface of the membrane assembly faces an inner space of the first culture tank; placing the first culture tank with the membrane assembly in a culture well of a first culture plate; culturing a first tissue cell on the first surface of the membrane assembly; removing the first culture tank with the membrane assembly from the culture well of the first culture plate; separating the membrane assembly from the first culture tank; installing the membrane assembly in a second culture tank, wherein a second surface opposite to the first surface faces an inner space of the second culture tank; placing the membrane assembly in a culture well of a first culture plate; culturing a first tissue cell on the first surface of the membrane assembly; removing the first culture tank with the membrane assembly from the culture well of the first culture plate; separating the membrane assembly from the first culture tank; installing the membrane assembly in a second culture tank, wherein a second surface opposite to the first surface faces an inner space of the second culture tank; placing the membrane assembly in a culture well of a first culture plate; The second culture tank is placed in the culture well of the second culture plate; and a second tissue cell is cultured on the second surface of the membrane assembly.

The foregoing has provided a relatively broad overview of the technical features of the present disclosure to facilitate a better understanding of the detailed description of the present disclosure set forth below. Other technical features that constitute the subject matter of the patent claims of the present disclosure are described below. Those skilled in the art will appreciate that the concepts and specific embodiments disclosed below can be readily utilized to modify or design other structures or processes to achieve the same objectives as those of the present disclosure. Those skilled in the art will also appreciate that such equivalent constructions do not depart from the spirit and scope of the present disclosure as defined in the appended patent claims.

The following disclosure provides a number of different embodiments or examples for implementing different features of the provided subject matter. Specific examples of components and configurations will be described below to simplify the disclosure. Of course, these are merely examples and are not intended to be limiting. For example, in the following description, "forming a first component above or on a second component" may include embodiments in which the first component and the second component are formed in direct contact, and may also include embodiments in which additional components may be formed between the first component and the second component so that the first component and the second component may not be in direct contact. In addition, the disclosure may repeat component symbols and/or letters in various examples. This repetition is intended for simplicity and clarity and does not, in itself, indicate a relationship between the various embodiments and/or configurations discussed.

Furthermore, for convenience, spatially relative terminology (e.g., "below," "beneath," "below," "above," "upper," and the like) may be used herein to describe the relationship of one element or component to another element or component, as depicted in the figures. Spatially relative terminology is intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. The device may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein may be interpreted accordingly.

This disclosure provides a novel cell co-culture method and system for in vitro simulating living tissues, such as those involved in percutaneous absorption, general vascular tissue, and cerebrovascular tissue. A key innovation of this system lies in the ability to culture different cell populations on both sides of a membrane. This two-sided approach allows for the simultaneous culture of multiple cell types, allowing them to interact through the membrane while maintaining their own unique environmental conditions.

Figure 1 is a schematic diagram of a double-sided multi-cell co-culture device 1 according to an embodiment of the present disclosure. As shown in Figure 1 , the double-sided multi-cell co-culture device 1 may include a first culture tank 11, a second culture tank 12, and a membrane assembly 13. Referring to Figure 1 , the first culture tank 11 may include a hollow, generally cylindrical body 110. Body 110 may include a first end 111 and a second end 112 opposite first end 111. In some embodiments of the present disclosure, body 110 has a tapered shape from second end 112 to first end 111. Furthermore, first end 111 of body 110 has a first opening 1110, and second end 112 has a second opening 1120 (see Figure 3B ). Both first opening 1110 and second opening 1120 communicate with the interior space 1100 of body 110. This design allows for efficient fluid exchange and cell growth within the culture tank, providing an ideal environment for cell interaction and nutrient flow. The tapered structure also facilitates easy removal and addition of culture medium, improving the overall usability of the device.

Furthermore, the second culture tank 12 may include a hollow, generally cylindrical body 120, which may include a first end 121 and a second end 122 opposite the first end 121. In some embodiments of the present disclosure, the body 120 has a tapered shape from the second end 122 to the first end 121. Furthermore, the first end 121 of the body 120 has a first opening 1210, and the second end 122 has a second opening 1220. Both the first opening 1210 and the second opening 1220 (see FIG. 4B ) communicate with the interior space 1200 of the body 120. This configuration ensures that the second culture tank 12 offers similar advantages as the first culture tank 11, including efficient hydrodynamics and an enhanced cell environment. The cylindrical and conical designs of the two culture tanks ensure they are easy to manufacture and maintain, promoting consistency in experimental conditions.

The membrane assembly 13 is configured to be mounted on the first end 111 of the main body 110 of the first culture tank 11. In other words, the membrane assembly 13 is configured for use with the first culture tank 11. When the membrane assembly 13 is mounted on the first end 111 of the main body 110 of the first culture tank 11, the membrane assembly 13 effectively closes the first opening 1110 of the main body 110. Similarly, the membrane assembly 13 is also configured to be mounted on the first end 121 of the main body 120 of the second culture tank 12. In other words, the membrane assembly 13 is configured for use with the second culture tank 12. When the membrane assembly 13 is mounted on the first end 121 of the main body 120 of the second culture tank 12, the membrane assembly 13 effectively closes the first opening 1210 of the main body 120. In addition, when the membrane assembly 13 is used in conjunction with the first culture tank 11, the side of the membrane assembly 13 facing the interior space 1100 of the main body 110 is different from the side of the membrane assembly 13 facing the interior space 1200 of the main body 120 when used in conjunction with the second culture tank 12.

Figure 2A is a schematic diagram of a membrane assembly 13 of a double-sided multi-cell co-culture device 1 according to an embodiment of the present disclosure. Figure 2B shows a schematic cross-sectional view of the membrane assembly 13 of the double-sided multi-cell co-culture device 1 according to an embodiment of the present disclosure. Referring to Figures 2A and 2B , the membrane assembly 13 may include an annular member 131 and a membrane 133. The annular member 131 has two opposing sides 1311 and 1312. Side 1311 of the annular member 131 may have two protrusions 1313, and side 1312 of the annular member 131 may be connected to the membrane 133. As shown in Figure 2B , the membrane 133 may cover side 1312 of the annular member 131. That is, surface 1331 of membrane 133 can be attached to side 1312 of annular member 131 and recessed relative to side 1311 of annular member 131. Furthermore, opposing surface 1332 of membrane 133 is generally flat. In some embodiments of the present disclosure, membrane 133 is bonded to annular member 131 via a non-adhesive fusion method. This structure ensures a stable and leak-proof assembly, which is crucial for maintaining the integrity of co-culture conditions. The non-adhesive fusion method also minimizes potential chemical interference from adhesives, ensuring a more biocompatible environment for cell culture.

Membrane 133 is configured to culture different cell populations on its two opposing surfaces 1331 and 1332, respectively. Membrane 133 may include a transparent porous membrane made of materials such as PET, PC, PTFE, PVDF, etc. The pore size of the transparent porous membrane may be between 200 nanometers and 10 microns, preferably 400 nanometers, 1 micron, 3 microns, or 8 microns. These different pore sizes allow selective permeability, supporting the growth and interaction of various cell types. In addition, one or more biopolymers may be grafted onto the two surfaces 1331 and 1332 of membrane 133 to adjust the hardness of the substrate surface with which cells or tissues come into contact. In some embodiments of the present disclosure, plasma treatment can be performed to graft one or more biopolymers onto both surfaces 1331 and 1332 of membrane 133. These biopolymers may include gamma-PGA, hyaluronic acid, collagen, chitin, chitosan, fibronectin, and/or poly-L-lysine. This grafting process enhances the biocompatibility and functional properties of the membrane, allowing researchers to precisely tailor the cellular microenvironment.

Figure 3A is a schematic diagram of a first culture tank 11 of a double-sided multi-cell co-culture device 1 according to an embodiment of the present disclosure. Figure 3B is another schematic diagram of the first culture tank 11 of the double-sided multi-cell co-culture device 1. Figure 3C shows a schematic cross-sectional view of the first culture tank 11 of the double-sided multi-cell co-culture device 1. As shown in Figures 3A, 3B, and 3C, the first culture tank 11 may have a hollow body 110 that is generally cylindrical and tapers from a second end 112 toward a first end 111. The second end of the body 110 may include a second opening 1120 that communicates with the interior space 1100 of the body 110. Furthermore, the body 110 may include a plurality of flanges 113 near the second end 112. Each flange 113 may have a protrusion 1131.

The first end 111 of the main body 110 may include a first opening 1110 communicating with the interior space 1100 of the main body 110. The first opening 1110 may include an inner periphery 1102. Furthermore, the interior space 1100 of the main body 110 may include an annular surface 1101 adjacent to the first opening 1110 and connected to the inner periphery 1102. Two grooves 1103 may be formed on the annular surface 1101, and two trenches 1105 may be formed on the inner surface 1107 of the interior space 1100, each connected to the grooves 1103. Referring to Figures 3A, 3B, and 3C, these trenches 1105 extend generally longitudinally from the annular surface 1101 toward the interior space 1100 of the main body 110.

With further reference to FIG. 4 , when the membrane assembly 13 is mounted on the first end 111 of the main body 110 of the first culture tank 11, the membrane assembly 13 can be inserted into the first opening 1110. The annular member 131 of the membrane assembly 13 can closely fit the inner periphery 1102 of the first opening 1110, ensuring a secure and leak-proof installation. Furthermore, the side surfaces 1311 of the annular member 131 can closely fit the annular surface 1101, and the protrusions 1313 of the annular member 131 can be respectively inserted into the grooves 1103, thereby securely fitting the membrane assembly 13 to the first end 111 of the main body 110 of the first culture tank 11. This precise alignment and secure installation are crucial to maintaining the integrity of the co-culture environment, preventing contamination and ensuring consistent experimental conditions. In addition, the surface 1331 of the membrane 133 can face the interior space 1100 of the main body 110, providing a suitable surface for cell attachment and growth; while the surface 1332 of the membrane 133 faces away from the interior space 1100 of the main body 110, thereby allowing the separation of different types of cells or compartments.

Figures 5A, 5B, and 5C illustrate the process of separating a membrane assembly 13 from a first culture tank 11 according to an embodiment of the present disclosure. Referring to Figure 5A, a tool 2 is provided. Tool 2 may include a cylindrical body 21, which may have two flanges 22. The flanges 22 extend generally longitudinally along the outer surface of the cylindrical body 21. Tool 2 facilitates safe and efficient removal of the membrane assembly without damaging the membrane or the culture tank. The flanges 22 ensure proper alignment and provide a fulcrum during removal.

5B , the cylindrical body 21 of the tool 2 is inserted into the interior space 1100 of the main body 110 of the first culture tank 11 through the second opening 1120. The rims 22 of the cylindrical body 21 are aligned with the grooves 1105 formed on the inner surface 1107 of the interior space 1100. This alignment is crucial for effective force transmission, ensuring that the tool properly engages the internal structure of the culture tank. Furthermore, the top 220 of the rims 22 contact the protrusions 1313 of the annular component 131 of the membrane assembly 13, which are inserted into the grooves 1103 of the annular surface 1101 of the main body 110. This contact allows the tool to accurately apply pressure to the protrusions 1313, promoting their disengagement.

Referring to FIG. 5C , the user continues to press downward on the first culture tank 11, further inserting the cylindrical body 21 of the tool 2 into the first opening 1110 of the main body 110. The top portion 220 of the flange 22 of the tool 2 pushes further against the side 1311 and/or protrusion 1313 of the annular portion 131 of the membrane assembly 13, thereby disengaging the protrusion 1313 from the groove 1103 of the annular surface 1101 of the main body 110. This action effectively separates the membrane assembly 13 from the first culture tank 11. This method ensures controlled and damage-free removal of the membrane assembly 13, which is critical to maintaining the integrity of the experimental device and the reusability of the components. The design of the tool 2 and the step-by-step operation process provide a reliable and reproducible method for removing the membrane assembly 13, thereby improving the practicality and ease of use of the double-sided multi-cell co-culture device 1.

Figure 6A is a schematic diagram of the second culture tank 12 of the double-sided multi-cell co-culture device 1 according to an embodiment of the present disclosure. Figure 6B is another schematic diagram of the second culture tank 12 of the double-sided multi-cell co-culture device 1. Figure 6C shows a schematic cross-sectional view of the second culture tank 12 of the double-sided multi-cell co-culture device 1. As shown in Figures 6A, 6B, and 6C, the second culture tank 12 may have a hollow body 120 that is generally cylindrical and tapers from a second end 122 toward a first end 121. The second end 122 of the body 120 may include a second opening 1220 that communicates with the interior space 1200 of the body 120. Furthermore, the body 120 may include a plurality of flanges 123 near the second end 122. Each flange 123 may have a protrusion 1231.

The first end 121 of the main body 120 may include a first opening 1210 communicating with the interior space 1200 of the main body 120. The first end 121 of the main body 120 may include a surface 1211 facing away from the interior space 1200 of the main body 120 and a surface 1212 facing toward the interior space 1200 of the main body 120. The surface 1212 of the first end 121 may include a generally annular groove 1214, which may surround the first opening 1210. In addition, the surface 1211 of the first end 121 may include a generally annular protrusion 1213, which may correspond to the groove 1214 on the surface 1212 and surround the first opening 1210. In addition, the periphery of the first opening 1210 may include two flanges 125. Each flange 125 may have a through hole 1250.

With further reference to FIG. 7 , when the membrane assembly 13 is mounted on the first end 121 of the main body 120 of the second culture tank 12, it fits into the groove 1214 of the first end 121 and is generally aligned with the first opening 1210. The side surface 1311 of the annular member 131 partially rests against the groove 1214 and the flange 125, while the protrusions 1313 of the annular member 131 are inserted into the through-holes 1250 of the flange 125, thereby securely coupling the membrane assembly 13 to the first end 121 of the main body 120 of the second culture tank 12. This design ensures a secure and leak-proof connection, which is crucial for maintaining distinct environments on both sides of the membrane during co-culture experiments.

Furthermore, the surface 1332 of the membrane 133 can face the interior space 1200 of the main body 120, providing an ideal surface for cell attachment and growth within the second culture tank 12. In contrast, the surface 1331 of the membrane 133 faces away from the interior space 1200, which is crucial for maintaining separation between different cell types or experimental conditions.

Furthermore, when the membrane assembly 13 is mounted on the first end 121 of the main body 120 of the second culture tank 12, a portion of the side surface 1311 of the annular member 131 may not be covered by the protrusion 1213 on the surface 1211 of the first end 121. This exposed portion of the annular member 131 extends along the perimeter of the first opening 1210, providing an easily accessible edge for facilitating removal and handling of the membrane assembly 13. This design feature ensures that the membrane assembly 13 is securely fixed while also allowing for easy removal when needed, enhancing the convenience and ease of use of the double-sided multi-cell co-culture device 1.

Figures 8A, 8B, 8C, and 8D illustrate the process of installing a membrane assembly 13 into a second culture tank 12 according to an embodiment of the present disclosure. Referring to Figure 8A , a tool 3 is provided. The tool 3 may include a generally hollow cylindrical body 31, characterized by an annular groove 310. The groove 310 may be located at the top of the cylindrical body 31 and surround the hollow space of the cylindrical body 31. The tool 3 can help accurately position and securely install the membrane assembly 13 into the second culture tank 12, ensuring proper alignment and minimizing the risk of damage during installation.

8B , membrane assembly 13 is inserted into groove 310 of cylindrical body 31 of tool 3, with side surface 1311 of annular member 131 and surface 1331 of membrane 133 facing upward. Furthermore, protrusion 1313 of annular member 131 extends beyond groove 310 of cylindrical body 31. This configuration ensures that membrane assembly 13 is securely fixed in place and correctly positioned for insertion into a culture tank.

8C , the cylindrical body 31 of the tool 3 is inserted into the interior space 1200 of the main body 120 of the second culture tank 12 through the second opening 1220. At this point, the membrane assembly 13 may be aligned with the first opening 1210 of the main body 120, and the protrusion 1313 of the annular member 131 of the membrane assembly 13 may be aligned with the through-hole 1250 on the flange 125 of the first opening 1210.

8D , the user continues to press downward on the second culture tank 12, further inserting the cylindrical body 31 of the tool 3 into the first opening 1210 of the main body 120. As the user applies pressure, the protrusion 1313 of the annular member 131 of the membrane assembly 13 is pushed into the through-hole 1250 of the flange 125 of the first opening 1210. This action securely engages the protrusion 1313 with the flange 125, ensuring that the membrane assembly 13 is securely attached to the first end 121 of the main body 120 of the second culture tank 12. This method ensures a secure and stable installation of the membrane assembly 13, which is critical for maintaining the experimental conditions required for accurate and reproducible co-cultivation experiments.

Figures 9A, 9B, and 9C are schematic diagrams illustrating the process of separating a membrane assembly 13 from a second culture tank 12 according to an embodiment of the present disclosure. Referring to Figure 9A , a tool 4 is provided. Tool 4 includes a hollow protrusion 41 having two grooves 42. Grooves 42 extend generally longitudinally along the outer surface of protrusion 41. Tool 4 is specifically designed to facilitate safe and efficient removal of membrane assembly 13 from second culture tank 12, ensuring that the membrane and culture tank are not damaged during the process.

9B , the second culture tank 12 is placed on the tool 4. The first opening 1210 of the body 120 of the second culture tank 12 is generally aligned with the protrusion 41, and the flange 125 of the first opening 1210 is aligned with the groove 42 of the protrusion 41. Furthermore, the portion of the surface 1211 of the annular member 131 of the membrane assembly 13 exposed at the first end 121 of the body 120 rests against the top of the protrusion 41. This alignment ensures that the tool 4 can be correctly positioned, thereby applying pressure to the membrane assembly in a controlled manner.

Referring to FIG. 9C , the user continues to press downward on the second culture tank 12. As pressure is applied, the top of the protrusion 41 of the tool 4 pushes against the annular member 131 of the membrane assembly 13. This action disengages the protrusion 1313 of the annular member 131 from the through-hole 1250 of the flange 125 of the first opening 1210. When the flange 125 slides into the groove 42 of the protrusion 41, the membrane assembly 13 is effectively separated from the second culture tank 12. This method ensures that the membrane assembly 13 can be removed cleanly and without damage, preserving the integrity of the membrane and culture tank for future reuse. The tool design and step-by-step procedure provide a reliable and easy-to-use method for disassembling co-culture devices, increasing their practicality and efficiency in a laboratory setting.

FIG10A is a schematic diagram of a first culture tray 5 according to an embodiment of the present disclosure. As shown in FIG10A , the first culture tray 5 includes a plurality of culture wells 50. In some embodiments, the first culture tray 5 includes 12 culture wells 50 arranged in a matrix. Each culture well 50 is designed to accommodate a first culture tank 11. This arrangement allows for simultaneous culturing of multiple samples under consistent conditions, which is crucial for high-throughput experiments and comparative studies. The design of the culture tray and culture wells ensures that each culture tank is securely fixed, providing a stable environment for cell growth and interaction.

FIG10B shows a first culture tank 11 with a membrane assembly 13 placed in a culture well 50 of a first culture tray 5. Referring to FIG10B , the main body 110 of the first culture tank 11 is inserted into the interior space 500 of the culture well 50. The flange 113 of the first culture tank 11 is supported by the upper surface of the first culture tray 5, ensuring that a certain distance is maintained between the membrane assembly 13, mounted on the first end 111 of the main body 110, and the bottom 501 of the culture well 50. This distance is important because it prevents the membrane from directly contacting the bottom of the culture well, which could affect cell growth and membrane function.

Furthermore, as previously described, the membrane assembly 13 is configured to effectively seal the first opening 1110 of the main body 110 of the first culture tank 11. This seal ensures that the interior space 1100 of the main body 110 of the first culture tank 11 is isolated from the interior space 500 of the culture wells 50 of the first culture tray 5. This separation is crucial for maintaining distinct experimental conditions within the culture tank and culture wells, enabling researchers to control and monitor the specific environment of cell culture. This design also facilitates easy access to the culture medium and cells within the culture wells, enhancing the overall ease of use and flexibility of the co-culture device in various experimental settings.

FIG11A is a schematic diagram of a second culture tray 6 according to one embodiment of the present disclosure. As shown in FIG11A , the second culture tray 6 includes a plurality of culture wells 60. In some embodiments, the second culture tray 6 includes six culture wells 60 arranged in a matrix. Each culture well 60 is designed to accommodate a second culture tank 12. This design enables researchers to conduct multiple experiments simultaneously under the same conditions, facilitating comparative analysis and high-throughput screening. The arrangement of the culture wells ensures that each culture tank is securely positioned, providing a stable and controllable environment for cell culture.

Figure 11B shows the second culture tank 12 with the membrane assembly 13 placed in the culture well 60 of the second culture tray 6. Referring to Figure 11B , the main body 120 of the second culture tank 12 is inserted into the interior space 600 of the culture well 60. The flange 123 of the second culture tank 12 is supported by the upper surface of the second culture tray 6, ensuring that a certain distance is maintained between the membrane assembly 13, mounted on the first end 121 of the main body 120, and the bottom 601 of the culture well 60. This distance is important because it prevents the membrane from directly contacting the bottom of the culture well, which could interfere with the cell culture process and affect experimental results.

Furthermore, as previously described, the membrane assembly 13 is configured to effectively seal the first opening 1210 of the main body 120 of the second culture tank 12. This seal ensures that the interior space 1200 of the main body 120 of the second culture tank 12 is isolated from the interior space 600 of the culture wells 60 of the second culture tray 6. This separation is crucial for maintaining distinct experimental conditions within the culture tank and culture wells, enabling researchers to control and monitor the specific environment of cell culture. This design also facilitates easy access to the culture medium and cells within the culture wells, enhancing the overall ease of use and flexibility of the co-culture device in various experimental settings.

Figures 12A, 12B, 12C, 12D, 12E, 12F, 12G, and 12H illustrate a cell culture method according to an embodiment of the present disclosure. Referring to Figure 12A , a first culture tank 11 with a membrane assembly 13 is placed in a culture well 50 of a first culture tray 5. In some embodiments of the present disclosure, twelve first culture tanks 11 with membrane assemblies 13 can be placed in each of the twelve culture wells 50 of the first culture tray 5. As previously described, when the membrane assembly 13 is mounted on the first end 111 of the main body 110 of the first culture tank 11, the membrane assembly 13 effectively closes the first opening 1110, with the surface 1331 of the membrane 133 of the membrane assembly 13 facing the interior space 1100 of the main body 110 of the first culture tank 11. Furthermore, the culture wells 50 can be filled with a cell culture medium 55.

Referring to FIG. 12B , first tissue cells 71 are cultured in the first culture tank 11. Referring to FIG. 12B , the main body 110 of the first culture tank 11 is inserted into the culture well 50 of the first culture tray 5. The first end 111 of the main body 110 of the first culture tank 11 is immersed in the cell culture medium 55, causing the entire membrane assembly 13 to also be immersed in the cell culture medium 55. The first tissue cells 71 can be cultured within the interior space 1100 of the main body 110 of the first culture tank 11 and can also grow on the surface 1331 of the membrane 133 of the membrane assembly 13. In other words, the first tissue cells 71 can be cultured in a horizontal position. In some embodiments of the present disclosure, the first group of cells 71 may include one or more cell types.

Furthermore, trans-epithelial electrical resistance (TEER) can be monitored during the culture process. As shown in Figure 12B , electrodes 57 and 58 are respectively placed within the interior space 1100 of the main body 110 of the first culture tank 11 and the interior space 500 of the culture well 50 of the first culture tray 5 , to measure the electrical resistance across the membrane assembly 13.

12C , after the first tissue cells 71 have grown and covered the surface 1331 of the membrane 133 of the membrane assembly 13, the first culture tank 11 can be removed from the culture well 50 of the first culture tray 5, and the membrane assembly 13 can be removed from the first end 111 of the main body 110 of the first culture tank 11. In some embodiments of the present disclosure, the membrane assembly 13 can be removed from the first end 111 of the main body 110 of the first culture tank 11 using the tool 2 and/or the method shown in FIG. 5A , FIG. 5B , and FIG. 5C .

Referring to FIG. 12D , the membrane assembly 13 is mounted on the first end 121 of the main body 120 of the second culture tank 12. In some embodiments of the present disclosure, the membrane assembly 13 can be mounted on the first end 121 of the main body 120 of the second culture tank 12 using the tool 3 and/or the method shown in FIG. 8A , FIG. 8B , FIG. 8C , and FIG. 8D . As previously described, when the membrane assembly 13 is mounted on the first end 121 of the main body 120 of the second culture tank 12, the membrane assembly 13 effectively closes the first opening 1210, with the surface 1332 of the membrane 133 of the membrane assembly 13 facing the interior space 1200 of the main body 120 of the second culture tank 12. Furthermore, the first tissue cells 71 can be retained on the surface 1331 of the membrane 133 of the membrane assembly 13.

12E , the second culture tank 12 with the membrane assembly 13 is placed in the culture well 60 of the second culture tray 6. In some embodiments of the present disclosure, six second culture tanks 12 with membrane assemblies 13 can be placed in the six culture wells 60 of the second culture tray 6, respectively. Furthermore, the culture wells 60 can be filled with a cell culture medium 65.

12F , second tissue cells 72 are cultured in the second culture tank 12. Referring to FIG12F , the body 120 of the second culture tank 12 is inserted into the culture well 60 of the second culture tray 6. The first end 121 of the body 120 of the second culture tank 12 is immersed in the cell culture medium 65, causing the entire membrane assembly 13 to also be immersed in the cell culture medium 65. The second tissue cells 72 can be cultured in the interior space 1200 of the body 120 of the second culture tank 12, and the second tissue cells 72 can be grown on the surface 1332 of the membrane 133 of the membrane assembly 13. At the same time, the first tissue cells 71 retained on the surface 1331 of the membrane 133 of the membrane assembly 13 can be cultured in the interior space 600 of the culture wells 60 of the second culture dish 6. In other words, the first tissue cells 71 and the second tissue cells 72 can be cultured in a horizontal position. In some embodiments of the present disclosure, the second tissue cells 72 may include one or more cell types. In some embodiments, the cell type of the second tissue cells 72 can be the same as or different from the cell type of the first tissue cells 71.

Furthermore, trans-epithelial electrical resistance (TEER) can be monitored during the culture process. As shown in Figure 12F , electrodes 67 and 68 are respectively placed in the interior space 1200 of the main body 120 of the second culture tank 12 and the interior space 600 of the culture well 60 of the second culture tray 6 to measure the electrical resistance across the membrane assembly 13.

In some embodiments of the present disclosure, the third tissue cells 73 can be cultured in the interior space 600 of the culture wells 60 of the second culture dish 6. As shown in FIG12G , the third tissue cells 73 can grow on the bottom 601 of the interior space 600 of the culture wells 60. In other words, the first tissue cells 71, the second tissue cells 72, and the third tissue cells 73 can all be cultured in a horizontal state. In some embodiments of the present disclosure, the third tissue cells 73 may include one or more cell types. In some embodiments, the cell type of the third tissue cells 73 can be the same as or different from the cell type of the first tissue cells 71 and/or the second tissue cells 72.

12H , after the second tissue cells 72 have grown and covered the surface 1332 of the membrane 133 of the membrane assembly 13, the second culture tank 12 can be removed from the culture well 60 of the second culture tray 6, and the membrane assembly 13 can be removed from the first end 121 of the main body 120 of the second culture tank 12. In some embodiments of the present disclosure, the membrane assembly 13 can be removed from the first end 121 of the main body 120 of the second culture tank 12 using the tool 4 and/or the method shown in FIG. 9A , FIG. 9B , and FIG. 9C .

This disclosure provides a double-sided multi-cell co-culture method and system that addresses significant limitations of traditional monolayer cell culture systems. Key technical features and their corresponding advantages are as follows: -Dual-sided culture capability: Technical Features: This disclosure allows for the simultaneous culture of multiple cell types on both sides of a transparent porous membrane. The membrane is plasma-treated and suitable for grafting biopolymers such as gamma-PGA, hyaluronic acid, collagen, chitin, chitosan, and fibroin. Advantages: This dual-surface culture method can simulate more complex biological environments and interactions between different cell types, making it suitable for advanced tissue engineering and in vitro models of tissues such as skin, blood vessels, and the blood-brain barrier. -Triple Cell Co-Culture: Technical Features: This system enables the simultaneous culture of up to three different cell populations in a horizontal arrangement, each of which may be composed of multiple cell types. Advantages: This feature enhances the simulation of physiological conditions by constructing multilayered tissue structures (such as endothelial and epithelial tissues), which is crucial for realistic in vitro models in drug testing, disease modeling, and tissue regeneration research. - Real-time TEER Monitoring: Technical Features: This system can be equipped with electrodes to monitor transcellular membrane potential (TEER) in real time during cell culture. Advantages: Continuous TEER measurement ensures the integrity of the cell monolayer and provides important data on barrier function, which is crucial for studying epithelial and endothelial barrier properties and evaluating the effects of drugs and other compounds on the cell layer. -Customizable Membrane Stiffness: Technical Features: By adjusting the type and concentration of the grafted biopolymer, the membrane stiffness can be tailored to the specific needs of different cell types. Advantages: This flexibility enables researchers to optimize the mechanical properties of the cell culture environment to match those of native tissue, thereby promoting cell growth and function and increasing the accuracy of experimental results. -Scalable and Modular Design: Technical Features: The device features a modular design with removable culture chambers and membrane components, facilitating easy assembly, disassembly, and customization of culture setups. Advantages: The system's modularity and scalability facilitate high-throughput screening and large-scale studies, making it a versatile tool for research and industrial applications in biotechnology and pharmaceuticals. -Versatile Applications: Technical Features: The present invention is capable of simulating a variety of physiological environments, including percutaneous absorption, general vascular conditions, and the cerebrovascular environment. Advantages: This versatility expands the system's applicability to a wide range of biomedical research areas, including drug delivery, toxicology, and tissue engineering.

In summary, the double-sided multi-cell co-culture method and device represent significant advancements over traditional monolayer culture systems. Its ability to culture a wide variety of cell types in a controlled, physiologically sound environment, coupled with its real-time monitoring and customizable features, makes it a powerful tool for advancing research and development in medicine and biotechnology.

As used herein, terms such as "first," "second," and "third" describe various elements, components, regions, layers, and/or sections, and these elements, components, regions, layers, and/or sections should not be limited by these terms. These terms may be used solely to distinguish one element, component, region, layer, or section from another. Unless the context clearly indicates otherwise, the terms "first," "second," and "third" as used herein do not imply a sequence or order.

As used herein, the terms "substantially," "substantially," "essentially," and "approximately" are used to describe and explain small variations. When used in conjunction with an event or circumstance, the terms can refer to instances in which the event or circumstance occurred exactly as well as instances in which the event or circumstance occurred very approximately. For example, when used in conjunction with a numerical value, the terms can refer to a range of variation of less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. For example, two values may be considered "substantially" the same or equal if the difference between them is less than or equal to ±10% of a mean of the values (e.g., less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%). For example, "substantially" parallelism may involve an angular variation of less than or equal to ±10° relative to 0°, such as less than or equal to ±5°, less than or equal to ±4°, less than or equal to ±3°, less than or equal to ±2°, less than or equal to ±1°, less than or equal to ±0.5°, less than or equal to ±0.1°, or less than or equal to ±0.05°. For example, "substantially" perpendicular may involve an angular variation of less than or equal to ±10° relative to 90°, such as less than or equal to ±5°, less than or equal to ±4°, less than or equal to ±3°, less than or equal to ±2°, less than or equal to ±1°, less than or equal to ±0.5°, less than or equal to ±0.1°, or less than or equal to ±0.05°.

In addition, amounts, ratios, and other numerical values are sometimes presented in a range format. It should be understood that such range format, for convenience and brevity, should be interpreted flexibly to include not only the numerical values expressly specified as limits of the range, but also all individual numerical values or sub-ranges within the range, as if each individual numerical value and sub-range were expressly specified.

Although the present disclosure has been described and illustrated with reference to specific embodiments thereof, such descriptions and illustrations do not limit the present disclosure. Those skilled in the art will understand that various changes and equivalents may be made without departing from the true spirit and scope of the present disclosure as defined by the appended claims. The illustrations may not be drawn to scale. Due to differences in manufacturing processes and tolerances, there may be differences between the artistic representations in the present disclosure and the actual devices. The present disclosure may have other embodiments not specifically described. This specification and the drawings should be regarded as illustrative and not restrictive. Modifications may be made to adapt a particular situation, material, composition of matter, method, or process to the object, spirit, and scope of the present disclosure. All such modifications are intended to be included within the scope of the appended claims. Although the methods disclosed herein are described with reference to specific operations performed in a specific order, it should be understood that these operations can be combined, subdivided, or rearranged to form equivalent methods without departing from the teachings of the present disclosure. Therefore, unless specifically indicated herein, the order and grouping of operations should not be considered as limitations of the present disclosure.

1: Double-sided multi-cell co-culture device 2: Tool 3: Tool 4: Tool 5: First culture tray 6: Second culture tray 11: First culture tank 12: Second culture tank 13: Module 21: Cylinder 22: Flange 31: Cylinder 41: Protrusion 42: Groove 50: Culture well 55: Cell culture medium 57: Electrode 58: Electrode 60: Culture well 65: Cell culture medium 67: Electrode 68: Electrode 71: Cell 72: Cell 73: Cell 110: Main body 111: First end 112: Second end 113: Flange 120: Main body 121: First end 122: Second end 123: Flange 125: Flange 131: Annular member 133: Membrane 310: Recess 500: Internal space 501: Bottom 600: Internal space 601: Bottom 1100: Internal space 1101: Annular surface 1102: Inner periphery 1103: Recess 1105: Groove 1107: Inner surface 1110: First opening 1120: Second opening 1131: Protrusion 1200: Internal space 1210: First opening 1211: Surface 1212: Surface 1213: Protrusion 1214: Recess 1220: Second opening 1231: Protrusion 1250: Through-hole 1311: Side 1312: Side 1313: Protrusion 1331: Surface 1332: Surface

FIG1 is a schematic diagram of a double-sided multi-cell co-culture device according to an embodiment of the present disclosure.

FIG2A is a schematic diagram of a membrane assembly of a double-sided multi-cell co-culture device according to an embodiment of the present disclosure.

FIG2B is a schematic cross-sectional view of a membrane assembly of a double-sided multi-cell co-culture device according to an embodiment of the present disclosure.

FIG3A is a schematic diagram of a first culture tank of a double-sided multi-cell co-culture device according to an embodiment of the present disclosure.

FIG3B is another schematic diagram of the first culture tank of the double-sided multi-cell co-culture device according to an embodiment of the present disclosure.

FIG3C is a schematic cross-sectional view of the first culture tank of the double-sided multi-cell co-culture device according to an embodiment of the present disclosure.

FIG4 is a schematic cross-sectional view of a first culture tank equipped with a membrane assembly according to an embodiment of the present disclosure.

5A, 5B, and 5C are schematic diagrams illustrating the operation of separating the membrane assembly from the first culture tank according to an embodiment of the present disclosure.

FIG6A is a schematic diagram of a second culture tank of a double-sided multi-cell co-culture device according to an embodiment of the present disclosure.

FIG6B is another schematic diagram of the second culture tank of the double-sided multi-cell co-culture device according to an embodiment of the present disclosure.

FIG6C is a schematic cross-sectional view of the second culture tank of the double-sided multi-cell co-culture device according to an embodiment of the present disclosure.

FIG7 is a schematic cross-sectional view of a second culture tank equipped with a membrane assembly according to an embodiment of the present disclosure.

8A, 8B, 8C, and 8D are schematic diagrams illustrating the process of installing a membrane assembly into a second culture tank according to an embodiment of the present disclosure.

9A, 9B, and 9C are schematic diagrams illustrating the operation of separating the membrane assembly from the second culture tank according to an embodiment of the present disclosure.

FIG10A is a schematic diagram of a first culture tray according to an embodiment of the present disclosure.

FIG10B is a schematic diagram showing a first culture tank and a membrane assembly placed in one of the culture wells of a first culture plate according to an embodiment of the present disclosure.

FIG11A is a schematic diagram of a second culture tray according to an embodiment of the present disclosure.

FIG11B is a schematic diagram showing a second culture tank and a membrane assembly placed in one of the culture wells of a second culture plate according to an embodiment of the present disclosure.

FIG12A, FIG12B, FIG12C, FIG12D, FIG12E, FIG12F, FIG12G and FIG12H show a cell culture method according to an embodiment of the present disclosure.

1: Double-sided multi-cell co-culture device

11: First culture tank

12: Second culture tank

13: Modules

110: Subject

111: First End

112: Second End

120: Subject

121: First End

122: Second End

1100: Interior space

1110: First opening

1200: Interior space

1210: First opening

Claims (20)

A double-sided multi-cell co-culture device comprises: A first culture tank comprising: A main body, which is generally cylindrical and has a first end and a second end opposite the first end; A first opening, which is located at the first end of the main body; and A second opening, which is located at the second end of the main body; A second culture tank comprising: A main body, which is generally cylindrical and has a first end and a second end opposite the first end; A first opening, which is located at the first end of the main body; and A second opening, which is located at the second end of the main body; A membrane assembly The membrane assembly is configured to be removably mounted on the first end of the main body of the first culture tank, and when the membrane assembly is mounted on the first end of the main body of the first culture tank, the membrane assembly is configured to substantially close the first opening of the main body of the first culture tank. The membrane assembly is configured to be removably mounted on the first end of the main body of the second culture tank, and when the membrane assembly is mounted on the first end of the main body of the second culture tank, the membrane assembly is configured to substantially close the first opening of the main body of the second culture tank. The double-sided multi-cell co-culture device of claim 1, wherein the membrane assembly comprises: an annular member configured to mate with a periphery of the first opening of the main body of the first culture tank and configured to mate with a periphery of the first opening of the main body of the second culture tank; a membrane attached to the annular member. The double-sided multi-cell co-culture device of claim 2, wherein the membrane comprises a transparent porous membrane, and wherein one or more biopolymers are grafted onto two opposite surfaces of the transparent porous membrane. The double-sided multi-cell co-culture device of claim 3, wherein the transparent porous membrane is made of a material selected from the group consisting of PET, PC, PTFE and PVDF. The double-sided multi-cell co-culture device of claim 3, wherein a pore size of the transparent porous membrane is selected from the group consisting of 400 nanometers, 1 micron, 3 microns, and 8 microns. The double-sided multi-cell co-culture device of claim 3, wherein the one or more biopolymers are selected from the group consisting of gamma-PGA, hyaluronic acid, collagen, chitin, chitosan, fibronectin, and poly-L-lysine. The double-sided multi-cell co-culture device of claim 2, wherein the membrane substantially covers a first side of the annular member, and the membrane has a first surface recessed relative to a second side of the annular member, the second side being opposite to the first side of the annular member. The double-sided multi-cell co-culture device of claim 7, wherein, when the membrane assembly is mounted in the first opening of the main body of the first culture tank, the first surface of the membrane faces an interior space of the main body of the first culture tank; and wherein, when the membrane assembly is mounted in the first opening of the main body of the second culture tank, the first surface of the membrane faces away from an interior space of the main body of the second culture tank. A double-sided multi-cell co-culture system comprises: a first culture tank; a second culture tank; a membrane assembly, wherein the membrane assembly is configured to be removably mounted on the first culture tank and also configured to be removably mounted on the second culture tank; a first culture plate having at least one culture well, wherein the at least one culture well of the first culture plate is configured to accommodate the first culture tank; and a second culture plate having at least one culture well, wherein the at least one culture well of the second culture plate is configured to accommodate the second culture tank; When the first culture tank and the membrane assembly are installed and placed in the at least one culture well of the first culture plate, a first surface of the membrane assembly faces an interior space of the first culture tank, and a second surface of the membrane assembly faces a bottom of the at least one culture well of the first culture plate. When the second culture tank and the membrane assembly are installed and placed in the at least one culture well of the second culture plate, the second surface of the membrane assembly faces an interior space of the second culture tank, and the first surface of the membrane assembly faces a bottom of the at least one culture well of the second culture plate. The double-sided multi-cell co-culture system of claim 9, wherein, when the first culture tank and the membrane assembly are mounted and placed in the at least one culture well of the first culture plate, the membrane assembly is configured to isolate the interior space of the first culture tank from the interior space of one of the at least one culture wells of the first culture plate; and wherein, when the second culture tank and the membrane assembly are mounted and placed in the at least one culture well of the second culture plate, the membrane assembly is configured to isolate the interior space of the second culture tank from the interior space of one of the at least one culture wells of the second culture plate. The double-sided multi-cell co-culture system of claim 9, wherein when the first culture tank and the membrane assembly are installed and placed in the at least one culture well of the first culture plate, the first surface of the membrane assembly is configured to culture a first tissue cell. The double-sided multi-cell co-culture system of claim 9, wherein when the second culture tank and the membrane assembly are installed and placed in the at least one culture well of the second culture plate, the second surface of the membrane assembly is configured to culture a second tissue cell. The double-sided multi-cell co-culture system of claim 9, wherein when the second culture tank and the membrane assembly are installed and placed in the at least one culture well of the second culture plate, the bottom of the at least one culture well of the second culture plate is configured to culture third tissue cells. A cell co-culture method comprises: installing a membrane assembly in a first culture tank, wherein a first surface of the membrane assembly faces an interior space of the first culture tank; placing the first culture tank with the membrane assembly in a culture well of a first culture plate; culturing a first tissue cell on the first surface of the membrane assembly; removing the first culture tank with the membrane assembly from the culture well of the first culture plate; separating the membrane assembly from the first culture tank; installing the membrane assembly in a second culture tank, wherein a second surface of the membrane assembly faces an interior space of the second culture tank, the second surface being opposite to the first surface; Placing the second culture tank with the membrane assembly in a culture well of a second culture plate; and culturing a second tissue cell on the second surface of the membrane assembly. The method of claim 14 further comprises: while culturing the second tissue cells on the second surface of the membrane assembly, simultaneously culturing a third tissue cell on a bottom surface of the culture well of the second culture plate. The method of claim 14, further comprising: measuring a resistance across the membrane assembly while culturing the first tissue cells on the first surface of the membrane assembly. The method of claim 14, further comprising: measuring a resistance across the membrane assembly while culturing the second tissue cells on the second surface of the membrane assembly. The method of claim 14 further comprises: after removing the first culture tank with the membrane assembly from the culture hole of the first culture plate, inserting a first tool into the inner space of the first culture tank and using the first tool to push the membrane assembly, thereby separating the membrane assembly from the first culture tank. The method of claim 14 further comprises: after separating the membrane assembly from the first culture tank, placing the membrane assembly in a groove formed in a top portion of a second tool with the first surface of the membrane assembly facing upward, and then pressing the second culture tank downward to the top portion of the second tool so that the top portion of the second tool is inserted into the interior space of the second culture tank, thereby mounting the membrane assembly on the second culture tank. The method of claim 14 further comprises: after culturing the second tissue cells on the second surface of the membrane assembly, removing the second culture tank with the membrane assembly from the culture well of the second culture plate; and aligning the membrane assembly with a protrusion of a third tool and pressing the second culture tank with the membrane assembly downward onto the protrusion of the third tool, thereby separating the membrane assembly from the second culture tank.