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

Abstract

To provide means for efficiently co-culturing multiple types of cells that require different microenvironments.SOLUTION: A double-sided multi-cell-type co-culture system and a cell culture method are provided. The double-sided multi-cell-type co-culture system includes a first culture tank 11, a second culture tank 12, a membrane assembly 13, a first culture plate, and a second culture plate. The membrane assembly is configured to be removably attached to the first culture tank and configured to be removably attached to the second culture tank. The first culture plate has at least one well, and at least one well of the first culture plate is configured to receive the first culture tank provided with the membrane assembly. The second culture plate has at least one well, and at least one well of the second culture plate is configured to receive the second culture tank provided with the membrane assembly.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates, inter alia, to a double-sided multi-species cell co-culture system and a method for culturing different cell populations. [Background technology]

[0002] In the field of cell culture, traditional methods often use monolayer cell culture systems, which are generally limited to culturing a single cell type at a time. These systems typically use culture dishes or flasks made from materials such as polystyrene to provide a surface for cell attachment and growth. While effective for basic cell culture, these systems have significant limitations when it comes to simulating more complex biological environments.

[0003] One of the major challenges with conventional cell culture techniques is the inability to efficiently co-culture multiple cell types that require different microenvironments. For example, anaerobic bacteria require an oxygen-free environment, while many mammalian cells, including epithelial and endothelial cells, require an oxygen-containing environment. Creating a system that can simultaneously maintain these different conditions has been a complex and costly endeavor.

[0004] Additionally, current cell culture systems often require extensive handling and manipulation of cultures, increasing the risk of contamination and variability in experimental results, which is particularly problematic for applications that require high precision and reproducibility, such as drug testing and disease modeling. Summary of the Invention

[0005] According to one exemplary embodiment of the present disclosure, a double-sided multi-species cell co-culture device includes a first culture tank, a second culture tank, and a membrane assembly. The first culture tank has a substantially cylindrical body having a first end and a second end opposite the first end, a first opening at the first end of the body, and a second opening at the second end of the body. The second culture tank has a substantially cylindrical body having a first end and a second end opposite the first end, a first opening at the first end of the body, and a second opening at the second end of the body. The membrane assembly is configured to be removably attached to the first end of the body of the first culture tank. When the membrane assembly is attached to the first end of the body of the first culture tank, the membrane assembly is configured to substantially seal the first opening of the body of the first culture tank. The membrane assembly is configured to be removably attached to the first end of the body of the second culture tank. When the membrane assembly is attached to the first end of the body of the second culture tank, the membrane assembly is configured to substantially seal the first opening of the body of the second culture tank.

[0006] According to another exemplary embodiment of the present disclosure, a double-sided multi-species 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 attached to the first culture tank and to be removably attached to the second culture tank. The first culture plate has at least one well, and the at least one well of the first culture plate is configured to receive the first culture tank. When the first culture tank is attached to the membrane assembly and received in the at least one well of the first culture plate, a first surface of the membrane assembly faces the interior space of the first culture tank and a second surface of the membrane assembly faces the bottom of the at least one well of the first culture plate. The second culture plate has at least one well, and the at least one well of the second culture plate is configured to receive the second culture tank. When the second culture tank is attached to the membrane assembly and received in the at least one well of the second culture plate, the second surface of the membrane assembly faces the interior space of the second culture tank, and the first surface of the membrane assembly faces the bottom of the at least one well of the second culture plate.

[0007] According to one exemplary embodiment of the present disclosure, a method for co-culturing cells comprises: installing a membrane assembly in a first culture tank, wherein a first surface faces an interior space of the first culture tank; placing the first culture tank having the membrane assembly in a well of a first culture plate; culturing a first group of cells on the first surface of the membrane assembly; removing the first culture tank having the membrane assembly from the 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 the interior space of the second culture tank; placing the second culture tank having the membrane assembly in a well of a second culture plate; and culturing a second group of cells on the second surface of the membrane assembly.

[0008] To facilitate understanding of the present disclosure, the following embodiments are provided in conjunction with illustrations; however, the accompanying drawings are provided for reference and explanation purposes only and are not intended to limit the scope of the present disclosure. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram of a double-sided multi-cell co-culture device according to an embodiment of the present disclosure. [Figure 2A] FIG. 2A is a schematic diagram of a membrane assembly of a double-sided multi-species cell co-culture device according to an embodiment of the present disclosure. [Figure 2B] FIG. 2B is a schematic cross-sectional view of a membrane assembly of a double-sided multi-cell-species co-culture device according to an embodiment of the present disclosure. [Figure 3A] FIG. 3A is a schematic diagram of a first culture tank of a double-sided multi-species cell co-culture device according to an embodiment of the present disclosure. [Figure 3B] FIG. 3B is another schematic diagram of a first culture tank of a double-sided multi-cell co-culture device according to an embodiment of the present disclosure. [Figure 3C] FIG. 3C is a schematic cross-sectional view of a first culture tank of a double-sided multi-cell-species co-culture device according to an embodiment of the present disclosure. [Figure 4] FIG. 4 is a schematic cross-sectional view of a first culture tank equipped with a membrane assembly according to an embodiment of the present disclosure. [Figure 5A] FIG. 5A is a schematic diagram of an operation for separating a membrane assembly from a first culture tank according to an embodiment of the present disclosure. [Figure 5B] FIG. 5B is a schematic diagram of an operation for separating the membrane assembly from the first culture tank according to an embodiment of the present disclosure. [Figure 5C] FIG. 5C is a schematic diagram of an operation for separating the membrane assembly from the first culture tank according to an embodiment of the present disclosure. [Figure 6A] FIG. 6A is a schematic diagram of a second culture tank of a double-sided multi-species cell co-culture device according to an embodiment of the present disclosure. [Figure 6B] FIG. 6B is another schematic diagram of a second culture tank of a double-sided multi-cell co-culture device according to an embodiment of the present disclosure. [Figure 6C] FIG. 6C is a schematic cross-sectional view of a second culture tank of a double-sided multi-cell co-culture device according to an embodiment of the present disclosure. [Figure 7] FIG. 7 is a schematic cross-sectional view of a second culture tank including a membrane assembly according to an embodiment of the present disclosure. [Figure 8A] FIG. 8A is a schematic diagram of the operation of equipping a membrane assembly with a second culture tank according to an embodiment of the present disclosure. [Figure 8B] FIG. 8B is a schematic diagram of the operation of equipping a membrane assembly with a second culture tank according to an embodiment of the present disclosure. [Figure 8C] FIG. 8C is a schematic illustration of the operation of equipping a membrane assembly with a second culture tank according to an embodiment of the present disclosure. [Figure 8D] FIG. 8D is a schematic illustration of the operation of equipping a membrane assembly with a second culture tank according to an embodiment of the present disclosure. [Figure 9A] FIG. 9A is a schematic diagram of an operation for separating the membrane assembly from the second culture tank according to an embodiment of the present disclosure. [Figure 9B]FIG. 9B is a schematic diagram of an operation for separating the membrane assembly from the second culture tank according to an embodiment of the present disclosure. [Figure 9C] FIG. 9C is a schematic diagram of an operation for separating the membrane assembly from the second culture tank according to an embodiment of the present disclosure. [Figure 10A] FIG. 10A is a schematic diagram of a first culture plate according to an embodiment of the present disclosure. [Figure 10B] FIG. 10B is a schematic diagram illustrating a first culture tank having a membrane assembly received in one of the wells of a first culture plate, according to an embodiment of the present disclosure. [Figure 11A] FIG. 11A is a schematic diagram of a second culture plate according to an embodiment of the present disclosure. [Figure 11B] FIG. 11B is a schematic illustration of a second culture tank having a membrane assembly received in one of the wells of a second culture plate, according to an embodiment of the present disclosure. [Figure 12A] FIG. 12A illustrates a method of culturing cells according to an embodiment of the present disclosure. [Figure 12B] FIG. 12B illustrates a method of culturing cells according to an embodiment of the present disclosure. [Figure 12C] FIG. 12C illustrates a method of culturing cells according to an embodiment of the present disclosure. [Figure 12D] FIG. 12D illustrates a method of culturing cells according to an embodiment of the present disclosure. [Figure 12E] FIG. 12E illustrates a method of culturing cells according to an embodiment of the present disclosure. [Figure 12F] FIG. 12F illustrates a method of culturing cells according to an embodiment of the present disclosure. [Figure 12G] FIG. 12G illustrates a method of culturing cells according to an embodiment of the present disclosure. [Figure 12H] FIG. 12H illustrates a method of culturing cells according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0010] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to illustrate certain aspects of the present disclosure. Of course, these are merely illustrative and not intended to be limiting. For example, in the following description, "forming a first feature above or on a second feature" may include embodiments in which the first and second features are formed or arranged in direct contact with each other, and may also include embodiments in which an additional feature is formed or arranged between the first and second features such that the first and second features are not in direct contact with each other. Additionally, the present disclosure may repeat reference numerals and / or letters in various examples. This repetition is for simplicity and clarity and does not, in itself, dictate a relationship between the various embodiments and / or configurations discussed.

[0011] Spatially relative terms such as "beneath," "below," "above," "over," "on," "upper," "lower," "left," "right," "vertical," "horizontal," "side," and the like may be used herein to describe the relationship of one element or feature to other element(s) or feature(s) as illustrated in the figures for ease of description. Spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation shown in the figures. The device may be in other orientations (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein will be interpreted accordingly. When an element is referred to as "connected to" or "coupled to" another element, it should be understood that the element may be directly connected or coupled to the other element, or that intervening elements may be present.

[0012] This disclosure provides a novel method and system for co-culturing different cell populations, which can be used to simulate in vitro biological tissues such as those involved in percutaneous absorption, general blood vessels, and cerebral blood vessels. One of the key innovations of this system is the ability to culture different cell populations on both sides of a membrane. This double-sided approach allows for the simultaneous culture of multiple cell types, allowing them to interact through the membrane while maintaining the specific environmental conditions for each cell.

[0013] FIG. 1 is a schematic diagram of a double-sided multi-species cell co-culture device 1 according to an embodiment of the present disclosure. As shown in FIG. 1, the double-sided multi-species cell co-culture device 1 may include a first culture tank 11, a second culture tank 12, and a membrane assembly 13. Referring to FIG. 1, the first culture tank 11 may include a hollow, substantially cylindrical body 110, which may include a first end 111 and a second end 112 opposite the first end 111. In some embodiments of the present disclosure, the body 110 tapers from the second end 112 toward the first end 111. Furthermore, the first end 111 of the body 110 has a first opening 1110, and the second end 112 of the body 110 has a second opening 1120 (see FIG. 3B ), both of which are in fluid communication with an interior space 1100 of the body 110. This design allows for efficient fluid exchange and cell growth within the reservoir, providing an optimal environment for cell interaction and nutrient flow. The tapered structure also aids in easy medium removal and addition, improving the device's overall usability.

[0014] Additionally, the second culture tank 12 may include a hollow, substantially 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 tapers from the second end 122 toward the first end 121. Furthermore, the first end 121 of the body 120 has a first opening 1210, and the second end 122 of the body 120 has a second opening 1220, both of which are in fluid communication with the interior space 1200 of the body 120 (see FIG. 4B ). This configuration ensures that the second culture tank 12 provides similar benefits to the first culture tank 11, including efficient fluid movement and an enhanced cell environment. The cylindrical and tapered design of both tanks facilitates manufacturing and maintenance and promotes consistency of experimental conditions.

[0015] The membrane assembly 13 is configured to be attached to the first end 111 of the body 110 of the first culture tank 11. That is, the membrane assembly 13 is configured to be installed in the first culture tank 11. When the membrane assembly 13 is attached to the first end 111 of the body 110 of the first culture tank 11, the membrane assembly 13 is configured to substantially seal the first opening 1110 of the body 110 of the first culture tank 11. The membrane assembly 13 is configured to be attached to the first end 121 of the body 120 of the second culture tank 12. That is, the membrane assembly 13 is configured to be installed in the second culture tank 12. When the membrane assembly 13 is attached to the first end 121 of the body 120 of the second culture tank 12, the membrane assembly 13 is configured to substantially seal the first opening 1210 of the body 120 of the second culture tank 12. Furthermore, the side of the membrane assembly 13 facing the internal space 1100 of the main body 110 when the membrane assembly 13 is installed in the first culture tank 11 is different from the side of the membrane assembly 13 facing the internal space 1200 of the main body 120 when the membrane assembly 13 is installed in the second culture tank 12.

[0016] FIG. 2A is a schematic diagram of a membrane assembly 13 of a double-sided multi-species cell co-culture device 1 according to an embodiment of the present disclosure. FIG. 2B is a schematic cross-sectional view of the membrane assembly 13 of a double-sided multi-species cell co-culture device 1 according to an embodiment of the present disclosure. Referring to FIGS. 2A and 2B, the membrane assembly 13 may include a ring 131 and a membrane 133. The ring 131 has two opposing side surfaces 1311 and 1312. The side surface 1311 of the ring 131 may have two protrusions 1313, and the side surface 1312 of the ring 131 may be attached to the membrane 133. As shown in FIG. 2B, the membrane 133 may cover the side surface 1312 of the ring 131. That is, the surface 1331 of the membrane 133 is attached to the side surface 1312 of the ring 131 and may have a concave shape relative to the side surface 1311 of the ring 131. Furthermore, the surface 1332 of the membrane 133 opposite the surface 1331 is substantially flat. In some embodiments of the present disclosure, the membrane 133 is joined to the ring 131 using a non-adhesive fusion method. This construction ensures a robust, leak-free assembly, which is crucial for maintaining the integrity of the co-culture conditions. The non-adhesive fusion method also minimizes potential chemical interference from adhesives, ensuring a more biocompatible environment for cell culture.

[0017] The membrane 133 is configured to culture different cell populations on two opposing surfaces 1331 and 1332, respectively. The membrane 133 can include a transparent porous membrane, which can be made of materials such as PET, PC, PTFE, or PVDF. The pore size of the transparent porous membrane can be 400 nm, 1 micron, 3 microns, or 8 microns. Varying the pore size allows for selective permeability and can support the growth and interaction of various cell types. Furthermore, one or more biopolymers can be grafted onto both surfaces 1331 and 1332 of the membrane 133 to adjust the stiffness of the substrate surface that contacts cells or tissues. In some embodiments of the present disclosure, one or more biopolymers can be grafted onto both surfaces 1331 and 1332 of the membrane 133 by plasma treatment. The one or more biopolymers can include γ-PGA, hyaluronic acid, collagen, chitin, chitosan, fibroin, and / or poly-L-lysine. This grafting process improves the membrane's biocompatibility and functional properties, allowing researchers to precisely tailor the cell microenvironment.

[0018] FIG. 3A is a schematic diagram of a first culture tank 11 of a double-sided multi-species cell co-culture device 1 according to an embodiment of the present disclosure. FIG. 3B is another schematic diagram of the first culture tank 11 of a double-sided multi-species cell co-culture device 1 according to an embodiment of the present disclosure. FIG. 3C is a schematic cross-sectional view of the first culture tank 11 of a double-sided multi-species cell co-culture device 1 according to an embodiment of the present disclosure. As shown in FIGS. 3A, 3B, and 3C, the first culture tank 11 can have a hollow body 110 that is substantially cylindrical and can taper from the second end 112 to the first end 111. The second end of the body 110 can have a second opening 1120 that is in fluid communication with the interior space 1100 of the body 110. Furthermore, the body 110 can have multiple flanges 113 adjacent to the second end 112 of the body 110. Each of the flanges 113 can have a protrusion 1131.

[0019] The first end 111 of the body 110 may include a first opening 1110 that is in fluid communication with an interior space 1100 of the body 110. The first opening 1110 may have an inner circumferential surface 1102. Further, the interior space 1100 of the body 110 may have a ring-shaped surface 1101 adjacent to the first opening 1110 and connected to the inner circumferential surface 1102. Two curved recesses 1103 may be formed in the ring-shaped surface 1101, and two grooves 1105 may be formed in an inner surface 1107 of the interior space 1100 and connected to the curved recesses 1103, respectively. Referring to FIGS. 3A, 3B, and 3C, the grooves 1105 may extend substantially longitudinally from the ring-shaped surface 1101 toward the interior space 1100 of the body 110.

[0020] Referring further to FIG. 4 , when the membrane assembly 13 is attached to the first end 111 of the body 110 of the first culture tank 11, the membrane assembly 13 can be received in the first opening 1110. The ring 131 of the membrane assembly 13 fits snugly against the inner circumferential surface 1102 of the first opening 1110, ensuring a secure and leak-free fit. Furthermore, the side surface 1311 of the ring 131 abuts against the ring-shaped surface 1101, and the protrusions 1313 of the ring 131 are inserted into the curved recesses 1103, respectively, allowing the membrane assembly 13 to firmly engage with the first end 111 of the body 110 of the first culture tank 11. This precise alignment and secure fit are crucial for maintaining the integrity of the co-culture environment, preventing contamination, and ensuring consistent experimental conditions. Furthermore, the surface 1331 of the membrane 133 faces the interior space 1100 of the body 110, providing a suitable surface for cell attachment and growth. The surface 1332 of the membrane 133 faces away from the interior space 1100 of the body 110, allowing for the separation of different cell types or compartments.

[0021] 5A, 5B, and 5C are schematic illustrations of an operation for separating a membrane assembly 13 from a first culture tank 11 according to an embodiment of the present disclosure. Referring to FIG. 5A, a tool 2 is provided. The tool 2 may include a cylinder 21, which may include two flanges 22. The flanges 22 extend substantially longitudinally along the outer surface of the cylinder 21. The tool 2 can facilitate safe and efficient removal of the membrane assembly without damaging either the membrane or the culture tank. The flanges 22 ensure proper alignment and leverage during the removal process.

[0022] Referring to FIG. 5B , the cylinder 21 of the tool 2 is inserted through the second opening 1120 into the interior space 1100 of the body 110 of the first culture tank 11. The flanges 22 of the cylinder 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 and ensures that the tool properly engages with the interior features of the culture tank. Furthermore, the upper portion 220 of the flange 22 contacts the protrusions 1313 of the ring 131 of the membrane assembly 13, which are inserted into the curved recesses 1103 on the ring-shaped surface 1101 of the body 110. This contact allows the tool to precisely apply pressure to the protrusions, facilitating their removal.

[0023] Referring to FIG. 5C , the user continues to press down on the first culture tank 11, further inserting the cylinder 21 of the tool 2 toward the first opening 1110 of the main body 110. The upper portion 220 of the flange 22 of the tool 2 further presses against the side surface 1311 and / or protrusion 1313 of the ring 131 of the membrane assembly 13, disengaging the protrusion 1313 from the curved recess 1103 of the ring-shaped 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, which is essential for maintaining the integrity of the experimental setup and ensuring the reusability of components. This tool design and step-by-step process provide a reliable and reproducible method for membrane assembly removal, improving the practicality and ease of use of the double-sided multi-species cell co-culture device.

[0024] FIG. 6A is a schematic diagram of the second culture tank 12 of the double-sided multi-species cell co-culture device 1 according to an embodiment of the present disclosure. FIG. 6B is another schematic diagram of the second culture tank 12 of the double-sided multi-species cell co-culture device 1 according to an embodiment of the present disclosure. FIG. 6C is a schematic cross-sectional view of the second culture tank 12 of the double-sided multi-species cell co-culture device 1 according to an embodiment of the present disclosure. As shown in FIGS. 6A, 6B, and 6C, the second culture tank 12 can have a hollow body 120 that is substantially cylindrical and can taper from the second end 122 to the first end 121. The second end 122 of the body 120 can have a second opening 1220 that is fluidly connected to the interior space 1200 of the body 120. Furthermore, the body 120 can include multiple flanges 123 adjacent to the second end 122 of the body 120. Each of the flanges 123 can have a protrusion 1231.

[0025] The first end 121 of the body 120 may include a first opening 1210 that is in fluid communication with the interior space 1200 of the body 120. The first end 121 of the body 120 may have a surface 1211 facing away from the interior space 1200 of the body 120 and a surface 1212 facing the interior space 1200 of the body 120. The surface 1212 of the first end 121 may have a substantially ring-shaped recess 1214, and the recess 1214 may surround the first opening 1210. Furthermore, the surface 1211 of the first end 121 may include a substantially ring-shaped protrusion 1213 formed on the surface 1212 and corresponding to the recess 1214 that surrounds the first opening 1210. Furthermore, the first opening 1210 may have two flanges 125 formed on the periphery of the first opening 1210. Each of the flanges 125 may have a through-hole 1250.

[0026] 7 , when the membrane assembly 13 is attached to the first end 121 of the body 120 of the second culture tank 12, the membrane assembly 13 can be received in the recess 1214 of the first end 121 and substantially aligned with the first opening 1210. The side surfaces 1311 of the ring 131 partially abut against the recess 1214 and the flange 125, and the protrusions 1313 of the ring 131 are inserted into the through-holes 1250 of the flange 125, respectively, allowing the membrane assembly 13 to securely engage with the first end 121 of the body 120 of the second culture tank 12. This design ensures a stable and leak-free connection, which is essential for maintaining different environments on either side of the membrane in co-culture experiments.

[0027] Additionally, surface 1332 of membrane 133 may face interior space 1200 of body 120, providing an optimal surface for cell attachment and growth within the second culture tank. Conversely, surface 1331 of membrane 133 may be facing away from interior space 1200, which is important for maintaining separation between different cell types and experimental conditions.

[0028] Furthermore, when the membrane assembly 13 is attached to the first end 121 of the body 120 of the second culture tank 12, a portion of the side surface 1311 of the ring 131 may not be covered by the protrusion 1213 on the surface 1211 of the first end 121. This exposed portion of the ring 131 may extend along the periphery of the first opening 1210, providing an accessible edge that facilitates easy removal and handling of the membrane assembly. This design feature ensures that the membrane is securely fastened while allowing it to be easily removed if necessary, improving the ease of use and practicality of the double-sided multi-species cell co-culture device.

[0029] 8A, 8B, 8C, and 8D are schematic diagrams of an operation for installing a membrane assembly 13 in a second culture tank 12 according to an embodiment of the present disclosure. Referring to FIG. 8A, a tool 3 is provided. The tool 3 can include a substantially hollow cylinder 31 characterized by a ring-shaped recess 310. The recess 310 can be formed in the top of the cylinder 31 and surround the hollow space of the cylinder 31. The tool 3 can facilitate accurate placement and secure installation of the membrane assembly 13 in the second culture tank 12, ensuring proper alignment and minimizing the risk of damage during installation.

[0030] 8B, the membrane assembly 13 is received within the recess 310 of the cylinder 31 of the tool 3 with the side surface 1311 of the ring 131 and the surface 1331 of the membrane 133 facing upward. Additionally, the protrusion 1313 of the ring 131 extends beyond the recess 310 of the cylinder 31. This configuration ensures that the membrane assembly 13 is held in place and oriented correctly when inserted into the culture tank.

[0031] 8C , the cylinder 31 of the tool 3 is inserted into the internal space 1200 of the body 120 of the second culture tank 12 through the second opening 1220. The membrane assembly 13 can be aligned with the first opening 1210 of the body 120, and the protrusion 1313 of the ring 131 of the membrane assembly 13 can be aligned with the through-hole 1250 of the flange 125 of the first opening 1210.

[0032] 8D , the user continues to press down on the second culture tank 12 to further insert the cylinder 31 of the tool 3 toward the first opening 1210 of the body 120. As the user applies pressure, the protrusion 1313 of the ring 131 of the membrane assembly 13 may be pressed into the through-hole 1250 of the flange 125 of the first opening 1210. This action firmly engages the protrusion 1313 with the flange 125, firmly attaching the membrane assembly 13 to the first end 121 of the body 120 of the second culture tank 12. This method ensures a secure and stable attachment of the membrane assembly, which is crucial for maintaining the experimental conditions necessary for accurate and reproducible co-culture experiments.

[0033] 9A, 9B, and 9C are schematic illustrations of an operation for separating a membrane assembly 13 from a second culture tank 12 according to an embodiment of the present disclosure. Referring to FIG. 9A, a tool 4 is provided. The tool 4 includes a hollow protrusion 41 characterized by two grooves 42. These grooves 42 extend substantially longitudinally along the outer surface of the protrusion 41. The tool 4 is specially designed to facilitate safe and efficient removal of the membrane assembly 13 from the second culture tank 12, ensuring that the membrane and tank are not damaged during the process.

[0034] 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 substantially aligned with the protrusion 41. The flange 125 of the first opening 1210 is aligned with the groove 42 of the protrusion 41. Furthermore, the portion of the ring 131 of the membrane assembly 13 exposed on the surface 1211 of the first end 121 of the body 120 abuts the top of the protrusion 41. This alignment ensures that the tool 4 is properly positioned to exert a force on the membrane assembly in a controlled manner.

[0035] Referring to Figure 9C, the user continues to press down on the second culture tank 12. As pressure is applied, the top of the protrusion 41 of the tool 4 presses against the ring 131 of the membrane assembly 13. This action disengages the protrusion 1313 from the through-hole 1250 in the flange 125 of the first opening 1210. As the flange 125 slides into the groove 42 of the protrusion 41, the membrane assembly 13 is effectively detached from the second culture tank 12. This method ensures clean and damage-free removal of the membrane assembly, preserving the integrity of both the membrane and the culture tank for future use. This tool design and step-by-step process provide a reliable, user-friendly method for disassembling co-culture devices, improving practicality and efficiency in laboratories.

[0036] FIG. 10A is a schematic diagram of a first culture plate 5 according to an embodiment of the present disclosure. As shown in FIG. 10A, the first culture plate 5 includes a plurality of wells 50. In some embodiments, the first culture plate 5 includes 12 wells arranged in a matrix. Each well 50 is configured to receive a first culture tank 11. This arrangement allows for the simultaneous culture of multiple samples under consistent conditions, which is essential for high-throughput experiments and comparative studies. The design of the culture plate and wells ensures that each culture tank is held securely in place, providing a stable environment for cell growth and interaction.

[0037] 10B shows that the first culture tank 11 having the membrane assembly 13 is received in one of the wells 50 of the first culture plate 5. Referring to FIG. 10B, the body 110 of the first culture tank 11 is inserted into the interior space 500 of the well 50. The flange 113 of the first culture tank 11 is supported on the upper surface of the first culture plate 5, such that the membrane assembly 13 attached to the first end 111 of the body 110 is spaced a certain distance from the bottom 501 of the well 50. This spacing is important to prevent direct contact between the membrane and the bottom of the well, which could otherwise affect cell growth and membrane function.

[0038] Furthermore, as previously described, the membrane assembly 13 is configured to substantially seal the first opening 1110 of the body 110 of the first culture tank 11. This seal ensures that the interior space 1100 of the body 110 of the first culture tank 11 is isolated from the interior space 500 of the well 50 of the first culture plate 5. This isolation is essential for maintaining different experimental conditions within the culture tanks and wells, allowing researchers to control and monitor specific environments for cell culture. This design also allows easy access to the culture medium and cells within the wells, improving the overall usability and flexibility of the co-culture device in various experimental setups.

[0039] FIG. 11A is a schematic diagram of a second culture plate 6 according to an embodiment of the present disclosure. As shown in FIG. 11A, the second culture plate 6 includes a plurality of wells 60. In some embodiments, the second culture plate 6 includes six wells arranged in a matrix. Each well 60 is configured to receive a second culture tank 12. This design allows researchers to simultaneously conduct multiple experiments under identical conditions, facilitating comparative analysis and high-throughput screening. The well arrangement ensures that each culture tank is securely positioned, providing a stable and controlled environment for cell culture.

[0040] 11B shows that the second culture tank 12 having the membrane assembly 13 is received in one of the wells 60 of the second culture plate 6. Referring to FIG. 11B, the body 120 of the second culture tank 12 is inserted into the interior space 600 of the well 60. The flange 123 of the second culture tank 12 is supported on the upper surface of the second culture plate 6, so that the membrane assembly 13 attached to the first end 121 of the body 120 is spaced a certain distance from the bottom 601 of the well 60. This spacing is important to prevent direct contact between the membrane and the bottom of the well, which could otherwise interfere with the cell culture process and affect experimental results.

[0041] Furthermore, as previously described, the membrane assembly 13 is configured to substantially seal the first opening 1210 of the body 120 of the second culture tank 12. This seal ensures that the interior space 1200 of the body 120 of the second culture tank 12 is isolated from the interior space 600 of the well 60 of the second culture plate 6. This isolation is essential for maintaining different experimental conditions within the culture tank and well, allowing researchers to control and monitor specific environments for cell culture. This design also allows easy access to the culture medium and cells within the well, improving the overall usability and flexibility of the co-culture device in various experimental setups.

[0042] 12A, 12B, 12C, 12D, 12E, 12F, 12G, and 12H illustrate a method for culturing cells according to an embodiment of the present disclosure. Referring to FIG. 12A, a first culture tank 11 having a membrane assembly 13 is received in one of the wells 50 of a first culture plate 5. In some embodiments of the present disclosure, twelve first culture tanks 11 equipped with membrane assemblies 13 can be individually placed in the twelve wells 50 of the first culture plate 5. As described above, when the membrane assembly 13 is attached to the first end 111 of the body 110 of the first culture tank 11, the membrane assembly 13 can substantially seal the first opening 1110, and the surface 1331 of the membrane 133 of the membrane assembly 13 can face the interior space 1100 of the body 110 of the first culture tank 11. Furthermore, the well 50 can be filled with a cell culture medium 55.

[0043] 12B, a first cell group 71 is cultured in a first culture tank 11. Referring to FIG. 12B, the body 110 of the first culture tank 11 is inserted into a well 50 of a first culture plate 5. A first end 111 of the body 110 of the first culture tank 11 is immersed in a cell culture solution 55, and the entire membrane assembly 13 is also immersed in the cell culture solution 55. The first cell group 71 can be cultured in the internal space 1100 of the body 110 of the first culture tank 11. The first cell group 71 can also be cultured on a surface 1331 of the membrane 133 of the membrane assembly 13. That is, the first cell group 71 can be cultured in a horizontal state. In some embodiments of the present disclosure, the first cell group 71 includes one or more types of cells.

[0044] Furthermore, the transepithelial electrical resistance (TEER) can be monitored during the culture process. As shown in Figure 12B, electrodes 57, 58 can be disposed in the inner space 1100 of the body 110 of the first culture tank 11 and the inner space 500 of the well 50 of the first culture plate 5, respectively, to measure the electrical resistance of the membrane assembly 13.

[0045] 12C , after the first cell population 71 has grown to cover the surface 1331 of the membrane 133 of the membrane assembly 13, the first culture tank 11 can be removed from the well 50 of the first culture plate 5, and the membrane assembly 13 can be removed from the first end 111 of the body 110 of the first culture tank 11. In some embodiments of the present disclosure, the membrane assembly 13 is removed from the first end 111 of the body 110 of the first culture tank 11 using the tool 2 and / or method shown in FIGS. 5A , 5B, and 5C.

[0046] 12D , the membrane assembly 13 is attached to the first end 121 of the body 120 of the second culture tank 12. In some embodiments of the present disclosure, the membrane assembly 13 is attached to the first end 121 of the body 120 of the second culture tank 12 using the tool 3 and / or method shown in FIGS. 8A , 8B , 8C , and 8D . As described above, when the membrane assembly 13 is attached to the first end 121 of the body 120 of the second culture tank 12, the membrane assembly 13 can substantially seal the first opening 1210, and the surface 1332 of the membrane 133 of the membrane assembly 13 can face the interior space 1200 of the body 120 of the second culture tank 12. Furthermore, the first cell group 71 can be retained on the surface 1331 of the membrane 133 of the membrane assembly 13.

[0047] 12E, the second culture tank 12 equipped with the membrane assembly 13 is received in one of the wells 60 of the second culture plate 6. In some embodiments of the present disclosure, six second culture tanks 12 equipped with the membrane assemblies 13 can be individually placed in six wells 60 of the second culture plate 6. Furthermore, the wells 60 can be filled with a cell culture medium 65.

[0048] 12F, a second cell group 72 is cultured in the second culture tank 12. Referring to FIG. 12F, the body 120 of the second culture tank 12 is inserted into the well 60 of the second culture plate 6. The first end 121 of the body 120 of the second culture tank 12 is immersed in the cell culture solution 65, and the entire membrane assembly 13 is also immersed in the cell culture solution 65. The second cell group can be cultured in the internal space 1200 of the body 120 of the second culture tank 12. The second cell group 72 can be cultured on the surface 1333 of the membrane 133 of the membrane assembly 13. Meanwhile, the first cell group 71 held on the surface 1331 of the membrane 133 of the membrane assembly 13 can be cultured in the internal space 600 of the well 60 of the second culture plate 6. That is, the first cell group 71 and the second cell group 72 can be cultured in a horizontal position. In some embodiments of the present disclosure, second cell population 72 includes one or more types of cells. In some embodiments of the present disclosure, the cell types of second cell population 72 may be the same as or different from the cell types of first cell population 71.

[0049] Furthermore, the transepithelial electrical resistance (TEER) can be monitored during the culture process. As shown in Figure 12F, electrodes 67, 68 can be disposed in the inner space 1200 of the body 120 of the second culture tank 12 and the inner space 600 of the well 60 of the second culture plate 6, respectively, to measure the electrical resistance of the membrane assembly 13.

[0050] In some embodiments of the present disclosure, the third cell group 73 can be cultured in the interior space 600 of the well 60 of the second culture plate 6. As shown in FIG. 12G, the third cell group 73 can be cultured on the bottom 601 of the interior space 600 of the well 60. That is, the first cell group 71, the second cell group 72, and the third cell group 73 can be cultured in a horizontal position. In some embodiments of the present disclosure, the third cell group 73 includes one or more types of cells. In some embodiments of the present disclosure, the type of cells in the third cell group 73 can be the same as or different from the type of cells in the first cell group 71 and / or the type of cells in the second cell group 72.

[0051] 12H, after the second cell population 72 has grown to cover the surface 1332 of the membrane 133 of the membrane assembly 13, the second culture tank 12 can be removed from the well 60 of the second culture plate 6, and the membrane assembly 13 can be removed from the first end 121 of the body 120 of the second culture tank 12. In some embodiments of the present disclosure, the membrane assembly 13 is removed from the first end 121 of the body 120 of the second culture tank 12 using the tool 4 and / or method shown in FIGS. 9A, 9B, and 9C.

[0052] The present disclosure provides a double-sided multi-species cell co-culture method and system that addresses significant limitations in conventional monolayer cell culture systems. The following are key technical features and corresponding advantages: -Double-sided incubation capacity: Technical Features: This disclosure enables the simultaneous culture of multiple cell types on both sides of a transparent porous membrane, which is plasma-treated to graft biopolymers such as gamma-PGA, hyaluronic acid, collagen, chitin, chitosan, and fibroin. Advantages: This dual-sided approach allows for the ability to simulate more complex biological environments and interactions between different cell types, making it suitable for advanced tissue engineering and in vitro modeling of tissues such as skin, blood vessels, and the brain barrier. -3 cell group co-culture: Technical Features: The system is capable of culturing up to three different cell populations (each population may consist of multiple cell types) in a horizontal array. Benefits: This capability enables the creation of multi-layered tissue constructs, such as endothelial and epithelial tissues, enhancing the simulation of physiological conditions, which is essential for realistic in vitro models for drug testing, disease modeling, and tissue regeneration studies. -Real-time TEER monitoring: Technical Features: The system can be equipped with electrodes that allow real-time monitoring of transepithelial electrical resistance (TEER) during the cell culture process. Benefits: Continuous measurement of TEER ensures the integrity of the cell monolayer and provides important data on barrier function, which is essential for studies involving epithelial and endothelial barrier properties and for assessing the effects of drugs and other compounds on the cell layer. -Customizable membrane stiffness: Technical Features: The stiffness of the membrane can be tuned by varying the type and concentration of grafted biopolymers to suit the specific requirements of different cell types. Benefits: This adaptability allows researchers to optimize the mechanical properties of the cell culture environment to match that of the native tissue, enhancing cell growth and function and improving the accuracy of experimental results. -Scalable and modular design: Technical Features: The device features a modular design with removable culture tank and membrane assemblies, allowing for easy assembly, disassembly, and customization of the culture setup. Advantages: The modularity and scalability of the system facilitates high-throughput screening and large-scale studies, making it a versatile tool for both research and industrial applications in biotechnology and medicine. -Various uses: Technical Features: The present disclosure can simulate various physiological environments, including transdermal absorption, general vascular conditions, and cerebrovascular conditions. Advantages: This versatility broadens the system's range of applications, making it suitable for a wide range of biomedical research areas, including drug delivery, toxicology, and tissue engineering.

[0053] In summary, the double-sided multi-cell co-culture method and device represents a significant advance over conventional monolayer culture systems. Its ability to culture multiple cell types in a controlled, physiologically relevant environment, coupled with its real-time monitoring and customizable features, positions it as a powerful tool for advancing research and development in the fields of medicine and biotechnology.

[0054] As used herein, the singular terms "a," "an," and "the" may include plural referents unless the context clearly dictates otherwise.

[0055] As used herein, the terms "approximately," "substantially," "substantial," and "about" are used to describe and explain slight variations. When used in conjunction with an event or circumstance, these terms can refer not only to instances in which the event or circumstance occurs exactly, but also to instances in which the event or circumstance occurs approximately. For example, when used in conjunction with a numerical value, these terms can refer to a range of variation of the numerical value of ±10% or less, such as ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%, or ±0.05%. For example, two numerical values ​​can be considered "substantially" identical or equal if the difference between the two numerical values ​​is ±10% or less of the mean value of the numerical values, such as ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%, or ±0.05%. For example, "substantially" parallel can refer to a range of angular variation from 0° of ±10° or less, e.g., ±5° or less, ±4° or less, ±3° or less, ±2° or less, ±1° or less, ±0.5° or less, ±0.1° or less, or ±0.05° or less. For example, "substantially" perpendicular can refer to a range of angular variation from 90° of ±10° or less, e.g., ±5° or less, ±4° or less, ±3° or less, ±2° or less, ±1° or less, ±0.5° or less, ±0.1° or less, or ±0.05° or less.

[0056] Additionally, quantities, ratios, and other numerical values ​​may be presented herein in a range format. It will be understood that such range format is used for convenience and brevity and should be understood flexibly to include not only the numerical values ​​expressly specified as the limits of the range, but also to include all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly specified.

[0057] While the present disclosure has been described and illustrated with reference to specific embodiments thereof, these descriptions and illustrations are not intended to limit the disclosure. Those skilled in the art should understand that various changes may be made and equivalents substituted without departing from the true spirit and scope of the present disclosure, as defined by the appended claims. Illustrations may not necessarily be drawn to scale. Manufacturing processes and tolerances may result in differences between the depictions in the drawings of this disclosure and the actual device. There may be other embodiments of the present disclosure not specifically illustrated. The specification and drawings are to be considered illustrative, not restrictive. Modifications may be made to adapt particular situations, materials, mixtures, methods, or processes to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto. Although methods disclosed herein are described with reference to particular operations performed in a particular order, it will be understood that these operations may be combined, sub-divided, or re-sequenced to form equivalent methods without departing from the teachings of the present disclosure. Accordingly, unless otherwise indicated herein, the order and grouping of operations is not intended to limit the disclosure.

Claims

1. a first culture tank having a substantially cylindrical body having a first end and a second end opposite the first end, a first opening provided at the first end of the body, and a second opening provided at the second end of the body; a second culture tank having a substantially cylindrical body having a first end and a second end opposite the first end, a first opening provided at the first end of the body, and a second opening provided at the second end of the body; a membrane assembly; the membrane assembly is configured to be removably attached to the first end of the body of the first culture tank, and when the membrane assembly is attached to the first end of the body of the first culture tank, the membrane assembly is configured to substantially seal the first opening of the body of the first culture tank; the membrane assembly is configured to be removably attached to the first end of the body of the second culture tank, and when the membrane assembly is attached to the first end of the body of the second culture tank, the membrane assembly is configured to substantially seal the first opening of the body of the second culture tank.

2. The membrane assembly comprises: a ring configured to fit around a periphery of the first opening of the body of the first culture tank and to fit around a periphery of the first opening of the body of the second culture tank; a membrane attached to the ring; The double-sided multi-cell co-culture device according to claim 1 , comprising:

3. The double-sided multi-cell co-culture device according to claim 2 , wherein the membrane comprises a transparent porous membrane, and one or more types of biopolymers are grafted onto both opposing surfaces of the transparent porous membrane.

4. The double-sided multi-species cell co-culture device according to claim 3 , wherein the transparent porous membrane is made of a material selected from the group consisting of PET, PC, PTFE, and PVDF.

5. 4. The double-sided multi-species cell co-culture device of claim 3, wherein the transparent porous membrane has a pore size selected from the group consisting of 400 nm, 1 micron, 3 microns, and 8 microns.

6. The double-sided multi-species cell co-culture device of claim 3 , wherein the one or more biopolymers are selected from the group consisting of γ-PGA, hyaluronic acid, collagen, chitin, chitosan, fibroin, and poly-L-lysine.

7. 3. The double-sided multi-species cell co-culture device of claim 2, wherein the membrane substantially covers a first side of the ring, the membrane having a first surface that is recessed relative to a second side of the ring, the second side being opposite the first side of the ring.

8. 8. The double-sided multi-species cell co-culture device according to claim 7, wherein when the membrane assembly is attached to the first opening of the main body of the first culture tank, the first surface of the membrane faces the interior space of the main body of the first culture tank, and when the membrane assembly is attached to the first opening of the main body of the second culture tank, the first surface of the membrane faces away from the interior space of the main body of the second culture tank.

9. a first culture tank; a second culture tank; a membrane assembly configured to be removably attached to the first culture tank and configured to be removably attached to the second culture tank; a first culture plate having at least one well, the at least one well of the first culture plate configured to receive the first culture tank; a second culture plate having at least one well, the at least one well of the second culture plate configured to receive the second culture tank; When the first culture tank is attached to the membrane assembly and received in the at least one 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 well of the first culture plate; When the second culture tank is attached to the membrane assembly and received in the at least one 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 well of the second culture plate.

10. 10. The double-sided multi-species cell co-culture system of claim 9, wherein when the first culture tank is attached to the membrane assembly and received in the at least one well of the first culture plate, the membrane assembly is configured to separate the interior space of the first culture tank from the interior space of the at least one well of the first culture plate, and when the second culture tank is attached to the membrane assembly and received in the at least one well of the second culture plate, the membrane assembly is configured to separate the interior space of the second culture tank from the interior space of the at least one well of the second culture plate.

11. 10. The double-sided multi-species cell co-culture system of claim 9, wherein the first surface of the membrane assembly is configured to culture a first group of cells when the first culture tank is attached to the membrane assembly and received in the at least one well of the first culture plate.

12. 10. The double-sided multi-species cell co-culture system of claim 9, wherein the second surface of the membrane assembly is configured to culture a second group of cells when the second culture tank is attached to the membrane assembly and received in the at least one well of the second culture plate.

13. 10. The double-sided multi-species cell co-culture system of claim 9, wherein when the second culture tank is attached to the membrane assembly and received in the at least one well of the second culture plate, the bottom of the at least one well of the second culture plate is configured to culture a third cell group.

14. installing a membrane assembly in a first culture tank, the membrane assembly having a first surface facing an interior space of the first culture tank; placing the first culture tank with the membrane assembly in a well of a first culture plate; culturing a first population of cells on the first surface of the membrane assembly; removing the first culture tank with the membrane assembly from the well of the first culture plate; separating the membrane assembly from the first culture tank; installing the membrane assembly in a second culture tank, the membrane assembly having a second surface opposite the first surface facing an interior space of the second culture tank; placing the second culture tank with the membrane assembly in a well of a second culture plate; and culturing a second population of cells on the second surface of the membrane assembly.

15. 15. The method of claim 14, further comprising culturing a third cell population on the bottom surface of the well of the second culture plate while culturing the second cell population on the second surface of the membrane assembly.

16. 15. The method of claim 14, further comprising measuring the electrical resistance of the membrane assembly while culturing the first cell population on the first surface of the membrane assembly.

17. 15. The method of claim 14, further comprising measuring the electrical resistance of the membrane assembly while culturing the second cell population on the second surface of the membrane assembly.

18. 15. The method of claim 14, further comprising, after removing the first culture tank with the membrane assembly from the well of the first culture plate, inserting a first tool into the interior 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.

19. 15. The method of claim 14, further comprising: after separating the membrane assembly from the first culture tank, placing the membrane assembly in a recess formed in an upper portion of a second tool with the first surface of the membrane assembly facing upward; and pressing the second culture tank downward against the upper portion of the second tool so that the upper portion of the second tool is inserted into the internal space of the second culture tank, thereby mounting the membrane assembly in the second culture tank.

20. After culturing the second cell population on the second surface of the membrane assembly, removing the second culture tank having the membrane assembly from the well of the second culture plate; 15. The method of claim 14, further comprising: aligning the membrane assembly with a protrusion of a third tool and pressing the second culture tank with the membrane assembly downward against the protrusion of the third tool, thereby separating the membrane assembly from the second culture tank.

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