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CN-116590146-B - Cell culture plate and application thereof in research of tunnel nanotubes formed between cells

CN116590146BCN 116590146 BCN116590146 BCN 116590146BCN-116590146-B

Abstract

The invention relates to the field of biological research, in particular to a cell culture plate and application thereof in researching tunnel nanotubes formed between cells. The cell culture plate comprises a substrate, wherein sample chambers are symmetrically arranged on the substrate, one side, close to each other, of each sample chamber is communicated through a connecting cavity, the connecting cavity comprises a porous vertical cavity, the connecting cavity is divided into an upper space and a lower space, the width of the porous vertical cavity is the same as that of the connecting cavity, the length range of the porous vertical cavity is 5-100 mu m, the height of the lower space is 1-2 mu m, TNTs generated between cells are allowed to pass through, cell bodies are not allowed to pass through, and therefore direct contact of the cell bodies in the two sample chambers is limited, and the lower space is a specific observation/research area of the TNTs. The invention can be used for researching tunnel nanotubes formed between cells and TNTs formed between two different cell populations.

Inventors

  • YOU YUE
  • ZHAO FENG
  • LI JINXIA

Assignees

  • 华南理工大学

Dates

Publication Date
20260505
Application Date
20230421

Claims (4)

  1. 1. The cell culture plate comprises a substrate, wherein sample chambers are arranged on the substrate, and are characterized in that each two sample chambers are symmetrically arranged, sampling holes are formed in the top ends of the outer sides of the sample chambers, the two sample chambers are in a side tapered gradually narrowing structure on the side close to each other, and the tail ends of the two sample chambers are connected together through a connecting cavity; the connecting cavity comprises a porous vertical cavity body, the connecting cavity body is divided into an upper space and a lower space, the upper space is a space inside the porous vertical cavity body, the lower space is a region between the bottom of the porous vertical cavity body and a substrate bottom plate, and the height of the lower space is 1 mu m; The top end of the porous vertical cavity is closed, and the top plate, the front side wall and the rear side wall of the porous vertical cavity are integrally connected with the cell culture plate, and the material of the porous vertical cavity is the same as that of the sample cavity; The width of the porous vertical cavity is the same as the width of the connecting cavity, and the length range of the porous vertical cavity is 5-100 mu m; the sample inlet end of the sample cavity is of a square structure, and the internal height of the sample cavity is 0.1mm; The inner side end of the sample chamber, namely from the initial part of the side cone to the connecting cavity, has the inner height of 0.2mm, and the bottom plate of the sample chamber at the part is overlapped with the bottom plate of the substrate.
  2. 2. The cell culture plate according to claim 1, wherein the filter membrane is any one of a polycarbonate membrane, a polyester fiber membrane and a polytetrafluoroethylene membrane, and the pore size is set to 0.4-3 μm.
  3. 3. The cell culture plate of claim 1, wherein the number of sample chambers provided on the substrate is an even number of 2 or more.
  4. 4. Use of a cell culture plate according to any one of claims 1 to 3 for studying tunnel nanotubes formed between cells, comprising: Respectively adding cell suspensions of two different types of cells into two symmetrical sample chambers, placing the sample chambers into a sterile CO 2 incubator for culturing, taking out the cell culture plate after the cells are attached, and placing the cell culture plate under an optical microscope for observing the lower space region of a connecting cavity; Or firstly, respectively marking F-actin of two different types of cells by using a fluorescent staining method, then, respectively adding cell suspensions of the two types of cells marked with different dyes into two symmetrical sample chambers, placing the sample chambers into a sterile CO 2 incubator for culturing, taking out the cell culture plate after the cells are adhered and stretched, observing the interaction of the F-actin between the two types of cells under a laser confocal microscope, and carrying out research on intercellular communication between the two types of cells mediated by a tunnel nano tube.

Description

Cell culture plate and application thereof in research of tunnel nanotubes formed between cells Technical Field The invention relates to the field of biological research, in particular to a cell culture plate and application thereof in researching tunnel nanotubes formed between cells. Background It has been found that, in addition to the action of some cytokines produced by autocrine or paracrine, intercellular communication can also form gap junctions and/or biological structures such as tunnel nanotubes (tunneling nanotubes, TNTs) that are in direct contact with each other to mediate information communication. The elongated membrane tubes formed from cell to cell are designated TNTs, and are also known as membrane nanotubes, nanotube-like structures, cytonemes, etc. TNTs are composed of cytoskeleton, are a connection mode for long-distance communication between cells, and can realize the transmission and exchange of intercellular organelles (such as mitochondria, golgi, and the like), cell membrane components, proteins, genetic materials, ions, and some small molecular materials. TNTs are used as a bridge to directly connect the monodisperse cells, so that deep cell communication is promoted. Numerous scientific studies have explored the in vivo and in vitro functions of TNTs, including but not limited to the effects of TNTs in substance transfer, signaling, cell differentiation and recoding, immune response, neurodegenerative disease, pathogen transmission, tumor development, and the like. TNTs have great scientific research significance and value as information communication mechanism. TNTs are free-grown cells, the structure (including direction, length and thickness) of the TNTs cannot be controlled or predicted by external force, the TNTs are staggered up and down and left and right, and finally form an interactive network or multi-layer stack, and the TNTs are thin-film tube nano-structures (the TNTs are generally smaller than 1 mu m in diameter and the cell body size is about 5 mu m or more) and cannot be observed by naked eyes. At present, in vitro studies on TNTs are to inoculate cells into a conventional culture vessel for culture, so that TNTs are naturally formed, therefore, whether by means of a laser confocal microscope or an electron microscope technology, the existence of TNTs is difficult to find and observe under a complicated and complicated growth background, and a cell culture vessel for controllably growing and forming TNTs in a specific area is lacking. Micro-nano machining technology refers to the optimized design, machining, assembly, system integration and application technology of components or systems composed of sub-millimeter, micron and nanometer scale elements. The micro-nano structure manufactured by the photoetching technology can obtain a final required structure or element by further etching or coating, the minimum result size and precision are generally determined by the resolution of photoetching or etching links, and in addition, the laser micro-machining technology converts high energy of laser into heat energy to etch away materials. Based on the technical problems of studying TNTs among cells and the development of the existing micro-nano processing technology, the invention aims to provide a cell culture plate, wherein a section of micrometer region is arranged between two sample chambers of the cell culture plate, so that TNTs with communication function among cells can be formed in the section of micrometer region in a controllable manner, and cell bodies are limited to pass through the region, thereby being convenient for imaging analysis, observation and study of the TNTs. Disclosure of Invention The invention aims to provide a cell culture plate and application thereof in researching tunnel nanotubes formed among cells. The technical scheme adopted is as follows: The cell culture plate comprises a substrate, wherein sample chambers are arranged on the substrate, and the cell culture plate is characterized in that each two sample chambers are symmetrically arranged, sample holes are formed in the top ends of the outer sides of the sample chambers, cell suspension can be added through the sample holes, the side, close to each other, of the two symmetrical sample chambers is in a structure with tapered sides, and the tail ends of the two symmetrical sample chambers are communicated through a connecting cavity; the connecting cavity comprises a porous vertical cavity, the porous vertical cavity divides the connecting cavity into an upper space and a lower space, the upper space is a space inside the porous vertical cavity, the lower space is a region between the bottom of the porous vertical cavity and a substrate bottom plate, and the height of the lower space of the connecting cavity is 1-2 mu m. Preferably, the material of the bottom plate of the lower space of the connecting cavity channel can be set to be high-permeability glass or pure plastic according t