A cell culture plate and its use in studying tunneling nanotubes formed between cells

Abstract

This invention relates to the field of biological research, specifically to a cell culture plate and its application in studying tunnel nanotubes formed between cells. The cell culture plate includes a substrate with sample chambers symmetrically arranged in pairs, connected by a connecting channel on their adjacent sides. The connecting channel comprises a porous vertical cavity dividing the channel into an upper and a lower space. The width of the porous vertical cavity is the same as the width of the connecting channel, and its length ranges from 5 to 100 μm. The height of the lower space is 1 to 2 μm, allowing TNTs formed between cells to pass through but not the cell bodies, thus restricting direct contact between cell bodies in the two sample chambers. The lower space serves as a specific observation / study area for the TNTs. This invention can be used to study tunnel nanotubes formed between cells, and also to study TNTs formed between two different cell populations.

Description

Technical Field

[0001] This invention relates to the field of biological research, specifically to a cell culture plate and its application in studying tunnel nanotubes formed between cells. Background Technology

[0002] Research has found that in addition to the role of autocrine or paracrine cytokines, intercellular communication also involves the formation of gap junctions and / or tunneling nanotubes (TNTs), direct contact biological structures that mediate information exchange. These elongated membrane tubes formed between cells are named TNTs, also known as membrane nanotubes, nanotube structures, or cytonemes. Composed of the cytoskeleton, TNTs are a form of long-distance intercellular communication, enabling the transfer and exchange of organelles (such as mitochondria and the Golgi apparatus), cell membrane components, proteins, genetic material, ions, and some small molecules. TNTs act as "bridges," directly connecting disparate cells and facilitating deeper cellular communication. Numerous scientific studies have explored the in vivo and in vitro functions of TNTs, including but not limited to their roles in substance transport, signal transduction, cell differentiation and recoding, immune responses, neurodegenerative diseases, pathogen transmission, and tumor development. TNTs, as an information exchange mechanism, have significant scientific research value.

[0003] TNTs grow freely from cells, and their structure (including orientation, length, and thickness) cannot be controlled or predicted by external forces. TNTs often form in an interwoven pattern, eventually creating an interactive network or multi-layered stack. Moreover, TNTs are thin-film tube nanostructures (TNTs are generally less than 1 μm in diameter, while cell bodies are approximately 5 μm or larger), making them invisible to the naked eye. Currently, in vitro studies on TNTs involve seeding cells in conventional culture dishes and allowing TNTs to form naturally. Therefore, it is difficult to find and observe the presence of TNTs in the complex growth background using either laser confocal microscopy or electron microscopy. There is a lack of cell culture dishes that allow for the controlled growth and formation of TNTs within specific areas.

[0004] Micro-nano fabrication technology refers to the optimized design, processing, assembly, system integration, and application of components and systems at the sub-millimeter, micrometer, and nanometer scales. This includes micro-nano fabrication based on photolithography, where contact lithography can achieve a resolution of 0.5 μm, while deep ultraviolet lithography can achieve 0.1 μm. Micro-nano structures fabricated using photolithography require further etching or coating to obtain the final desired structure or component. The minimum final size and precision are typically determined by the resolution of the photolithography or etching process. Additionally, there is laser micromachining technology, which converts the high energy of lasers into thermal energy to etch away materials.

[0005] Based on the technical challenges of intercellular TNTs mentioned above and the development of existing micro-nano fabrication technologies, the purpose of this invention is to provide a cell culture plate in which a micrometer-sized region is set between the two sample chambers of the cell culture plate, so that TNTs that play a communication role between cells can be formed in a relatively controllable manner within this micrometer-sized region, while restricting the passage of cell bodies through this region, which facilitates imaging analysis and observation of TNTs. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a cell culture plate and its application in studying tunnel nanotubes formed between cells. The technical solution adopted is as follows:

[0007] A cell culture plate includes a substrate with sample chambers on the substrate. The sample chambers are arranged symmetrically in pairs. Each sample chamber has an inlet hole at the top of its outer side, through which cell suspension can be added. The two symmetrical sample chambers have a side cone-shaped structure that gradually narrows on the side that is close to each other, and the ends are connected by a connecting channel.

[0008] The connecting channel includes a porous vertical cavity, which divides the connecting channel into an upper space and a lower space. The upper space is the space inside the porous vertical cavity; the lower space is the area between the bottom of the porous vertical cavity and the base plate of the substrate. The height of the lower space of the connecting channel is 1~2μm.

[0009] Preferably, the material of the bottom plate of the lower space of the connecting cavity can be set as high-transparency glass or pure plastic according to experimental requirements.

[0010] Preferably, the top of the porous vertical cavity is closed, and its top plate and front and rear side walls are integrally connected to the cell culture plate. The material of the top plate and front and rear side walls is the same as that of the sample chamber. The entire cell culture plate can be made of polystyrene or other plastic materials, or high-transparency glass, which can be transparent or black and opaque (for light protection). The bottom plate of the sample chamber, which is the cell adhesion surface, is treated with TC (tissue cultured) surface treatment to facilitate cell adhesion and growth. The left and right side walls and bottom plate of the porous vertical cavity are porous filter membrane structures, allowing the mutual flow of cell culture medium between the two symmetrical sample chambers.

[0011] Preferably, the filter membrane includes, but is not limited to, polycarbonate membrane, polyester fiber film, polyethylene terephthalate membrane, and polytetrafluoroethylene membrane, with a pore size set to 0.4~3μm.

[0012] Preferably, the width of the porous vertical cavity is the same as the width of the connecting channel, and the length of the porous vertical cavity ranges from 5 to 100 μm.

[0013] Preferably, the sample inlet end of the sample chamber has a square structure and its internal height is 0.1 mm.

[0014] Preferably, the height of the inner end of the sample chamber, i.e., from the initial part of the side cone to the connecting channel, is 0.2 mm. The bottom plate of the sample chamber at this part coincides with the bottom plate of the substrate. Generally, the height of the inner end of the sample chamber should be greater than the height of the inlet end so that there are enough cells to form TNTs in the lower space of the connecting channel.

[0015] Preferably, the number of sample chambers provided on the substrate is an even number greater than or equal to 2.

[0016] The application of a cell culture plate in studying tunnel nanotubes formed between cells, and the specific application method using the aforementioned cell culture plate includes:

[0017] Cell suspensions of two different cell types were added to two symmetrical sample chambers and cultured in a sterile CO2 incubator. After the cells adhered to the walls, the cell culture plate was removed and observed under an optical microscope in the lower space of the connecting cavity.

[0018] Alternatively, F-actin of two different cell types can be labeled using fluorescent staining. Then, cell suspensions of the two cell types labeled with different dyes can be added to two symmetrical sample chambers and cultured in a sterile CO2 incubator. After the cells adhere and spread, the cell culture plate can be removed and the interaction of F-actin between the two cell types can be observed under a laser confocal microscope to study the intercellular communication mediated by tunnel nanotubes between the two cell types.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] This invention is a cell culture plate specifically designed for studying tunnel nanotubes (TNTs) formed between cells. Two sample chambers are connected by a connecting channel, the lower space of which is limited to a height of 1-2 μm. Due to the height / thickness difference between the size of the cell body and the diameter of the TNTs, the 1-2 μm height allows the TNTs generated between cells to pass through but does not allow the cell bodies to pass through, preventing direct contact between the cell bodies in the two symmetrical sample chambers. The TNTs are restricted to growing in this region, thus making this region a specific observation / research area for TNTs. Attached Figure Description

[0021] Figure 1 These are the top view and side sectional view of the present invention.

[0022] Figure 2 This is an enlarged schematic diagram of the cells in Example 1 forming TNTs in the connecting cavities of the present invention.

[0023] In the figure, 1-substrate, 2-sample chamber, 3-sample inlet, 4-connecting channel, 5-porous vertical cavity. Detailed Implementation

[0024] The accompanying drawings are for illustrative purposes only; to better illustrate this embodiment, some components in the drawings may be omitted, enlarged, or reduced, and do not represent the actual dimensions of the product; terms such as "upper," "lower," "top," "bottom," "side," "outer," "horizontal," and "vertical," indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the drawings and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as limiting the invention. For those skilled in the art, some well-known structures and their descriptions in the drawings may be omitted, and therefore should not be construed as limiting the invention.

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will now be described in further detail with reference to the accompanying drawings.

[0026] Example 1

[0027] like Figure 1 As shown, a cell culture plate includes a substrate 1 with two sample chambers 2 symmetrically arranged. Each sample chamber 2 has an inlet 3 at its outermost tip. A suspension of mouse breast cancer cells 4T1 is added to one sample chamber 2 through the inlet 3, while primary CD8+ isolated and purified from the spleen of 8-week-old female BALB / c mice is added to the other sample chamber 2. + The T cell suspensions, with a concentration of 1×10⁻⁶ cells in each suspension. 4 Cells / mL. The sample inlet end of sample chamber 2 has a square structure, while the opposite side has a tapered structure that gradually narrows, finally forming a connecting channel 4 at the end, connecting the two sample chambers 2.

[0028] The connecting cavity 4 includes a porous vertical cavity 5, which divides the connecting cavity 4 into an upper space and a lower space. The upper space is the internal space of the porous vertical cavity 5. The lower space is the area between the bottom of the porous vertical cavity 5 and the bottom plate of the substrate 1. The height of this area is 1 μm, that is, the height of the lower space of the connecting cavity 4 is 1 μm.

[0029] The top of the porous vertical cavity 5 is closed. The top plate and front and rear sidewalls of the porous vertical cavity 5 are integrally connected to the cell culture plate. The material of the top plate and the front and rear sidewalls is the same as that of the sample chamber 2, which is polystyrene plastic. The entire cell culture plate is also made of polystyrene and is transparent. The bottom plate of the sample chamber 2 is the cell adhesion surface, which undergoes TC surface treatment (tissue cultured treated) to facilitate cell adhesion and growth. The left and right sidewalls and bottom plate of the porous vertical cavity 5 are made of polyethylene terephthalate filter membranes with a pore size of 0.4 μm, allowing the culture medium for 4T1 cells and CD8 in both sample chambers 2 to pass through. + The culture medium for T cells is exchanged between each other.

[0030] The width of the porous vertical cavity 5 is the same as the width of the connecting channel 4, and the length of the porous vertical cavity 5 is 50 μm.

[0031] As a further preferred design, the internal height of the sample chamber 2 at the inlet end is 0.1 mm; the internal height of the other end of the sample chamber 2, i.e., the inner end, from the initial part of the side cone to the connecting channel 4, is 0.2 mm, and the bottom plate of the sample chamber at this part coincides with the bottom plate of the substrate. Generally, the height of the inner end of the sample chamber 2 is greater than the height of the inlet end, so that there is sufficient amount of cells to form TNTs in the lower space of the connecting channel 4.

[0032] like Figure 2As shown, the vaccine was inoculated with 4T1 and CD8. + The T-cell culture plate was placed in a sterile CO2 incubator and cultured at 37°C, 5% CO2, and 95% relative humidity. After 7 hours, the cell culture plate was removed, and appropriate amounts of complete cell culture medium were added to the two sample chambers 2 through the sample inlet 3. The plate was then placed under an inverted microscope, and bright-field imaging was performed on the TNTs in the lower space region connecting the cavity 4. This indicates that the TNTs formed between breast cancer cells and immune cells are involved in intercellular communication.

[0033] Example 2

[0034] Four sample chambers 2 are provided on the substrate 1, and each pair of symmetrical sample chambers 2 are connected by a connecting channel 4. 4T1 of cell suspension (1×10⁻⁶) is added to each of the four sample chambers 2 through the sample inlet 3. 4 Cells / mL. The left and right sidewalls and bottom plate of the porous vertical cavity 5 are made of polytetrafluoroethylene filter membrane with a pore size of 1 μm, allowing the culture medium of 4T1 cells in each pair of symmetrical sample chambers 2 to flow between each other. The cells will sink due to gravity and eventually adhere to the bottom of the region at height b inside the sample chamber 2. Therefore, the TNTs formed between the cells will not pass through the pores of the porous vertical cavity 5 (see Figure 2 The lower space of the connecting cavity 4 has a height of 2μm, and the bottom plate in this area is made of high-transparency glass. The length of the porous vertical cavity 5, i.e., the connecting cavity 4, is 100μm.

[0035] Other areas not mentioned are the same as in Example 1.

[0036] Cell culture plates containing 4T1 cells in each of the four sample chambers 2 were placed in a sterile CO2 incubator at 37°C, 5% CO2, and 95% relative humidity. After 5 hours, the cell culture plates were removed, and the old culture medium in the four sample chambers 2 was removed using a pipette. 1X CellMask™ DeepRed Actin Tracking Stain and mAb against α-tubulin were added to each pair of symmetrical sample chambers to label actin and microtubules. The lower space connecting chamber 4 was then imaged under a laser confocal microscope to investigate the structural components of TNTs formed between the 4T1 cells.

[0037] Example 3

[0038] First, human mammary fibroblast (HMF) cell suspension was obtained by trypsin digestion. The HMF cells were resuspended in pre-warmed (37°C) 0.025 μM MitoTracker Green FM dye and incubated at 37°C for 30 min. After staining, the HMF@Mito cell pellet was obtained by centrifugation at 1000 rpm. The cells were then washed twice with PBS and resuspended in fresh complete culture medium. The cells were then seeded into one of the sample chambers 2 on substrate 1 (cell suspension concentration 1 × 10⁻⁶). 4 (cells / mL), the number of sample chambers 2 provided on the substrate 1 is two. A cell suspension of human breast cancer cells MCF7 is seeded into the other symmetrical sample chamber 2 through another inlet 3. The concentration of the cell suspension is 1×10⁻⁶. 4 Cells / mL. The left and right sidewalls and bottom plate of the porous vertical cavity 5 are made of polycarbonate filter membrane with a pore size of 3μm, allowing the culture medium for MCF7 cells and HMF cells in the sample chamber 2 to flow between each other. The length of the porous vertical cavity 5, i.e., the connecting channel 4, is 25μm, and the height of the lower space of the connecting channel 4 is 1.5μm. The bottom plate of this area is made of high-transparency glass.

[0039] The cell culture plates, inoculated with HMF@Mito cells and MCF7 cells respectively in two sample chambers 2, were placed in a sterile CO2 incubator under the following conditions: 37°C, 5% CO2, and relative humidity (95%). After 10 hours, the cell culture plates were removed, and the old culture medium in both sample chambers 2 was removed using a pipette. Subsequently, 1X CellMask™ Deep Red Actin Tracking Stain was added to both sample chambers 2 to label the F-actin of HMF@Mito cells and MCF7 cells. After staining, the lower space connecting cavity 4 was observed under a laser confocal microscope, revealing that MCF7@Actin&Mito, i.e., MCF7 cells exhibited co-localization of F-actin and mito, thus exploring whether human breast fibroblasts provide mitochondria for breast cancer cells.

[0040] Other areas not mentioned are the same as in Example 1.

[0041] In practical applications, the sample chambers 2 of the cell culture plate can be set to an even number of 2, 4, 6, 8, 10, 12... Each pair of adjacent chambers can be connected by a connecting channel 4. Through advanced and precise manufacturing technology, the height of the lower space of the connecting channel 4 is ensured to be 1~2μm. The bottom material of this area can be set to high-transparency glass or pure plastic according to experimental requirements.

[0042] In the description of this specification, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0043] In the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0044] Other areas not mentioned herein adopt the common practices in existing technologies and will not be detailed here.

[0045] Furthermore, it should be understood that although this specification describes the embodiments, it does not mean that the present invention only includes the technical solutions in the embodiments. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art, which are also within the protection scope of the present invention.

Claims

1. A cell culture plate, comprising a substrate, wherein a sample chamber is provided on the substrate, characterized in that, Two sample chambers are symmetrically arranged, and an inlet is provided at the top of the outer side of each sample chamber. The two sample chambers have a side cone shape that gradually narrows on the side that is close to each other, and the ends are connected together by a connecting channel. The connecting channel includes a porous vertical cavity, which divides the connecting channel into an upper space and a lower space. The upper space is the space inside the porous vertical cavity, and the lower space is the area between the bottom of the porous vertical cavity and the base plate of the substrate. The height of the lower space is 1μm. The top of the porous vertical cavity is closed, and its top plate, front and rear side walls are integrally connected with the cell culture plate, and its material is the same as that of the sample chamber; the left and right side walls and bottom plate of the porous vertical cavity are porous filter membrane structures. The width of the porous vertical cavity is the same as the width of the connecting channel, and the length of the porous vertical cavity ranges from 5 to 100 μm. The sample inlet of the sample chamber has a square structure with an internal height of 0.1 mm. The inner end of the sample chamber, from the initial part of the side cone to the connecting channel, has a height of 0.2 mm. The bottom plate of the sample chamber at this part coincides with the bottom plate of the substrate.

2. The cell culture plate according to claim 1, characterized in that, The filter membrane is any one of polycarbonate membrane, polyester fiber film, or polytetrafluoroethylene membrane, with a pore size set to 0.4~3μm.

3. A cell culture plate according to claim 1, characterized in that, The number of sample chambers provided on the substrate is an even number greater than or equal to 2.

4. The application of a cell culture plate in studying tunnel nanotubes formed between cells, using a cell culture plate according to any one of claims 1 to 3, characterized in that, Application methods include: Cell suspensions of two different cell types were added to two symmetrical sample chambers and cultured in a sterile CO2 incubator. After the cells adhered to the walls, the cell culture plate was removed and observed under an optical microscope in the lower space of the connecting cavity. Alternatively, F-actin of two different cell types can be labeled using fluorescence staining. Then, cell suspensions of the two cell types labeled with different dyes can be added to two symmetrical sample chambers and cultured in a sterile CO2 incubator. After the cells adhere and spread, the cell culture plate can be removed and the interaction of F-actin between the two cell types can be observed under a laser confocal microscope to study intercellular communication between the two cell types mediated by tunnel nanotubes.

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