CN112481109A - Visualization device and method for studying cellular chemotaxis - Google Patents
Visualization device and method for studying cellular chemotaxis Download PDFInfo
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Abstract
本发明属于体外细胞培养技术领域，涉及一种用于研究细胞氧趋化性的可视化装置和方法。该可视化装置包括：可视化的第一壳体、与第一壳体的第一端连通的第二壳体和与第一壳体的第二端连通的第三壳体；第一壳体的高度小于第二壳体的高度和第三壳体的高度，从而使第一壳体相对于第二壳体和第三壳体形成凹陷部；第二壳体具有第一进气口、第一加样口和第一出气口；第三壳体具有第二进气口、第二加样口和第二出气口；第一进气口和第二进气口分别位于第二壳体和第三壳体的侧部；第一出气口和第二出气口分别靠近第一进气口和第二进气口设置。通过该可视化装置能够利用“过桥涵洞”原理简单快捷地证实细胞对某种气体是否具有趋化性，并且成本低廉。
The invention belongs to the technical field of in vitro cell culture, and relates to a visualization device and method for studying the oxidative chemotaxis of cells. The visualization device includes: a visualized first shell, a second shell in communication with the first end of the first shell, and a third shell in communication with the second end of the first shell; the height of the first shell It is smaller than the height of the second shell and the height of the third shell, so that the first shell forms a recess relative to the second shell and the third shell; the second shell has a first air inlet, a first plus The sample port and the first air outlet; the third housing has a second air inlet, a second sample adding port and a second air outlet; the first air inlet and the second air inlet are located in the second housing and the third air inlet respectively. The side part of the casing; the first air outlet and the second air outlet are respectively arranged close to the first air inlet and the second air inlet. The visualization device can use the "bridge culvert" principle to easily and quickly confirm whether cells have chemotaxis to a certain gas, and the cost is low.
Description
Technical Field
The invention belongs to the technical field of in-vitro cell culture, and particularly relates to a visualization device and method for researching cell chemotaxis.
Background
In vitro cell subculture has become an important laboratory technique indispensable to modern medicine, modern medicine and biology. Previous researches show that some cells have electrochemotaxis and have the capacity of directional migration and directional proliferation under an electric field; certain cells are photo-chemotactic, have directional receptors for visible or invisible light within the cell, and exhibit intracellular physiological changes. Recent studies have shown that some cells have magnetic chemotaxis. For certain cells, such as normal mammalian epithelial cells, oxygen supply is required for their growth and development. Mammalian epithelial cells primarily utilize oxygen and hemoglobin dissolved in blood. Whether the epithelial cells of mammals have oxygen chemotaxis is a hypothesis at present, and visual experimental evidence proves that the normal epithelial cells have oxygen chemotaxis, namely the normal epithelial cells directionally migrate or proliferate to the areas with high dissolved oxygen gradient. Unlike normal epithelial cells, most malignant cells of epithelial origin cannot directly undergo energy metabolism through the tricarboxylic acid cycle due to prolonged ischemic hypoxia, but can only produce energy through anaerobic glycolysis, i.e., the Warburg effect, and are likely to exhibit no oxygen chemotaxis.
In the study of cellular oxygen chemotaxis abroad, local oxygen gradient difference formed by microtubules or microbubbles is mainly applied, and the local oxygen gradient difference needs to be controlled by matching with a corresponding microelectronic single chip microcomputer to form a complex multilayer composite culture plate, so the cost is very high, and the large-scale popularization is not facilitated. And the local oxygen gradient difference formed by the microtubules or the microbubbles is used for researching the damage of carbon monoxide to the neuron cells when the gas category is changed, for example, oxygen is changed into carbon monoxide, and the device needs to be redesigned, thereby increasing the expansion cost. More importantly, the existence of microtubule or microbubble system physically affects the migration of adherent cells, thereby generating systematic errors.
Therefore, there is a need for a device and method for simply and rapidly verifying whether a cell has oxygen chemotaxis.
Disclosure of Invention
The invention aims to provide a visualization device and a visualization method for researching cell chemotaxis, which are convenient for observing and judging whether cells have chemotaxis on gas to be researched.
In order to achieve the above object, a first aspect of the present invention provides a visualization device for studying chemotaxis of cells, the visualization device being a closed device; the visualization device includes: the device comprises a first shell, a second shell and a third shell, wherein the first shell is visualized, the second shell is communicated with a first end of the first shell, and the third shell is communicated with a second end of the first shell; the height of the first shell is smaller than that of the second shell and that of the third shell, so that the first shell forms a recess relative to the second shell and the third shell;
the second shell is provided with a first air inlet, a first sample adding port and a first air outlet; the third shell is provided with a second air inlet, a second sample adding port and a second air outlet; the first air inlet and the second air inlet are respectively positioned at the side parts of the second shell and the third shell and are close to the surfaces of the second shell and the third shell which are higher than the first shell; the first air outlet and the second air outlet are respectively arranged close to the first air inlet and the second air inlet; the first sample adding port and the second sample adding port are respectively positioned at the end parts of the second shell and the third shell.
In the present invention, the first housing, the second housing, and the third housing are all in a regular shape, and each have horizontal upper and lower surfaces. Therefore, the height of the first housing refers to the minimum distance from the lower surface to the upper surface of the first housing; the height of the second shell refers to the minimum distance from the lower surface to the upper surface of the second shell; the height of the third housing means a minimum distance from a lower surface to an upper surface of the third housing.
In the invention, the heights of the positions of the first air inlet, the first air outlet, the second air inlet and the second air outlet are higher than the height of the first shell, so as to prevent a culture solution added into the visualization device from blocking the first air inlet, the first air outlet, the second air inlet and the second air outlet, and meanwhile, a dissolved gas concentration gradient difference is formed in the culture solution in the first shell due to different volume concentrations of a gas to be studied above the liquid level of the second shell and the third shell, specifically, a dissolved oxygen concentration gradient difference is formed in the culture solution in the first shell due to different oxygen volume concentrations above the liquid level of the second shell and the third shell.
The person skilled in the art may choose a transparent material for the first housing to enable visualization of the first housing. Preferably, the first shell, the second shell and the third shell are made of transparent materials, so that an operator can observe the progress of the cell chemotaxis experiment in the visualization device conveniently. The transparent material may include: at least one of polyvinyl chloride (PVC), Polystyrene (PS), polymethyl methacrylate (commonly known as organic glass), methyl methacrylate (commonly known as acryl), and polycarbonate.
According to the present invention, preferably, the first air inlet, the first air outlet, the second air inlet and the second air outlet are located on the same horizontal plane.
The shape and configuration of the first, second and third housings of the visualization device may be determined empirically by one skilled in the art. For convenience of manufacture and experimental observation, the first shell may preferably have a rectangular structure. The non-connecting ends of the second shell, the third shell and the first shell are respectively of a necking structure which gradually narrows down, the first sample adding port and the second sample adding port are respectively arranged on the necking structure, and the first sample adding port and the second sample adding port are sealed by sealing covers.
In a second aspect, the present invention provides a method for studying chemotaxis of cells using the visualization device provided in the first aspect of the present invention, the method comprising the steps of:
s1, adding a culture solution containing cells to be researched into the visual device from the first sample adding port and/or the second sample adding port, filling the first shell with the culture solution containing the cells to be researched, wherein the liquid levels of the culture solution containing the cells to be researched in the second shell and the third shell are respectively lower than the first air inlet, the second air inlet, the first air outlet and the second air outlet and higher than the first shell;
s2, introducing mixed gas containing low-concentration gas to be researched into the second shell through the first gas inlet, and exhausting gas through the first gas outlet; introducing mixed gas containing high-concentration gas to be researched into the third shell through the second gas inlet, and exhausting gas from the second gas outlet at the same time to form concentration gradient difference for dissolving the gas to be researched in the culture solution containing the cells to be researched in the first shell;
and S3, observing whether the cell to be researched moves towards the direction of the second shell or the direction of the third shell through the first shell.
According to the invention, the gas mixture preferably contains, in addition to the gas to be investigated, carbon dioxide and nitrogen. In the present invention, carbon dioxide is used to provide the desired pH for cell culture, and is preferably 5%. The concentration of nitrogen varies with oxygen. Preferably, in a mixed gas containing a low concentration of the gas to be investigated, the concentration by volume of carbon dioxide may be 5% and the concentration by volume of nitrogen may be 94% to 93%. In a mixed gas containing a high concentration of the gas to be investigated, the volume concentration of carbon dioxide is 5% and the volume concentration of nitrogen is 73% -75%.
According to the invention, preferably, the gas to be investigated is oxygen.
According to the present invention, preferably, the concentration of the gas required for chemotaxis of the cells to be studied can be determined by a person skilled in the art through experiments, and when the gas to be studied is oxygen, the mixed gas containing the gas to be studied in a low concentration is a mixed gas containing oxygen in a low concentration, and the volume concentration of oxygen is 0.1% to 5%; the mixed gas containing high-concentration gas to be researched is mixed gas containing high-concentration oxygen, and the volume concentration of the oxygen is 15-25%; in a preferred embodiment of the invention, the volume concentration of oxygen in the mixed gas is 0.5% to 2%, preferably 1%; the volume concentration of oxygen in the mixed gas containing high-concentration oxygen is 20-22%, and the volume concentration of oxygen is preferably 21%.
It will be understood by those skilled in the art that, when the gas to be studied is oxygen, the mixed gas containing oxygen at a low concentration forms a low oxygen concentration region in the second enclosure, the mixed gas containing oxygen at a high concentration forms a high oxygen concentration region in the third enclosure, and an oxygen solubility gradient is formed in the culture solution in the first enclosure, and if the cell to be studied has chemotaxis for oxygen, the cell to be studied moves into the third enclosure through the culture solution in the first enclosure; conversely, the direction of migration of the cells to be investigated may be non-directional.
In the present invention, in particular, the cells to be investigated include epithelial cells. More particularly, the epithelial cells are mammalian epithelial cells.
According to the visualization device for researching cell chemotaxis provided by the invention, the culture solution containing the cells to be researched is added into the visualization device from the first sample adding port and/or the second sample adding port, the first shell is filled with the culture solution containing the cells to be researched, and the liquid levels of the culture solution containing the cells to be researched in the second shell and the third shell are respectively lower than the first air inlet and the second air inlet and higher than the first shell; introducing mixed gas containing low-concentration oxygen into the second shell through the first air inlet, and simultaneously introducing mixed gas containing high-concentration oxygen into the third shell through the second air inlet; if the cell to be studied has oxygen tropism, it can be observed whether the cell to be studied moves towards the third shell through the first shell, so as to determine whether the cell to be studied has oxygen tropism. Therefore, the visualization device can simply and quickly verify whether the cells to be researched have chemotaxis on oxygen by utilizing the 'bridge culvert' principle, and has low cost.
The composition of the culture medium can be selected by those skilled in the art according to the characteristics of the cells to be tested, and the present invention is not described herein in detail.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings, in which like reference numerals generally represent like parts throughout.
Fig. 1 shows a schematic view of a visualization device for studying cellular chemotaxis provided by the present invention.
Fig. 2 shows a state diagram of the use of a visualization device for studying cellular chemotaxis.
FIGS. 3 and 4 show graphs of the results of the study of cell chemotaxis in a single-cell animal of the invention using the device of the invention and an amoeba.
Fig. 5 shows a physical diagram of the visualization device for studying cellular chemotaxis provided by the present invention.
Description of the reference numerals
1-a first housing; 2-a second housing; 201-a first air inlet; 202-a first sample port; 203-a first air outlet; 3-a third shell; 301-a second air inlet; 302-a second sample port; 303-second air outlet.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
The following describes the visualization device for studying cellular chemotaxis provided by the present invention in detail with reference to fig. 1 and 5.
In the present invention, the visualization device for studying cellular chemotaxis is used to perform oxygen gradient differential culture on a single-cell amoeba animal in a living cell workstation (DeltaVision, Germany) to obtain a dissolved concentration gradient of the gas to be studied, as shown in FIG. 2, wherein the arrow pointing from the third shell to the second shell indicates that the concentration gradient differential of the gas to be studied is formed in the culture solution containing the cells to be studied in the first shell. And the first shell was photographed, obtaining fig. 3 and 4, the curves in fig. 3 and 4 being the trajectories of amoeba (analyzed by ImageJ). Fig. 5 shows a physical diagram of the visualization device for studying cellular chemotaxis provided by the present invention.
Example 1
The present embodiments provide a visualization device for studying cellular chemotaxis. Referring to fig. 1 and 5, the visualization device includes: the device comprises a visual first shell 1, a second shell 2 communicated with a first end of the first shell 1 and a third shell 3 communicated with a second end of the first shell 1; the height of the first casing 1 is smaller than the height of the second casing 2 and the height of the third casing 3, so that the first casing 1 forms a recess relative to the second casing 2 and the third casing 3; the second shell 2 is provided with a first air inlet 201, a first sample adding port 202 and a first air outlet 203; the third housing 3 has a second air inlet 301, a second sample addition port 302 and a second air outlet 303; the first air inlet 201 and the second air inlet 301 are located at the sides of the second casing 2 and the third casing 3, respectively, and are close to the surfaces of the second casing 2 and the third casing 3 higher than the first casing 1; the first air outlet 203 and the second air outlet 303 are respectively arranged close to the first air inlet 201 and the second air inlet 301; the first sample addition port 202 and the second sample addition port 302 are respectively positioned at the end parts of the second shell 2 and the third shell 3; the first casing 1, the second casing 2, and the third casing 3 are made of a transparent material. The non-connection ends of the second housing 2 and the third housing 3 and the first housing 1 are respectively a necking structure which gradually narrows, and the first sample port 202 and the second sample port 302 are respectively arranged on the necking structure. The first shell 1 is of a rectangular structure; both the first sample port 202 and the second sample port 302 are sealed with a sealing lid. The first air inlet 201, the first air outlet 203, the second air inlet 301 and the second air outlet 303 are located on the same horizontal plane.
Example 2
This example provides a method for studying cellular chemotaxis using the visualization device of example 1. The cell to be studied is a single-celled animal of amoeba, the method comprising the steps of:
s1, adding a culture solution containing cells to be researched into the visual device from the first sample adding port 202 and the second sample adding port 302, filling the first shell 1 with the culture solution containing the cells to be researched, wherein the liquid levels of the culture solution containing the cells to be researched in the second shell 2 and the third shell 3 are respectively lower than the first air inlet 201, the second air inlet 301, the first air outlet 203 and the second air outlet 303 and higher than the first shell 1;
s2, introducing mixed gas containing low-concentration oxygen into the second shell 2, and exhausting through the first air outlet 203; introducing mixed gas containing high-concentration oxygen into the third shell 3 through the second gas inlet 301, and exhausting gas through the second gas outlet 303 to form a dissolved oxygen concentration gradient difference in a culture solution containing cells to be researched in the first shell 1;
s3, observing whether the cell to be researched moves towards the direction of the third shell 3 through the first shell 1.
As shown in fig. 3 and 4, the migration of the cell to be studied towards the third housing 3, i.e. the migration of the cell to be studied towards the hyperoxia region, indicates that the cell to be studied has oxygen chemotaxis towards the gas to be studied.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Claims (9)
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030022362A1 (en) * | 2000-11-08 | 2003-01-30 | Gregory Kirk | Cell motility and chemotaxis test device and methods of using same |
| CN103080294A (en) * | 2010-09-08 | 2013-05-01 | 株式会社岛津制作所 | Cell culture container and cell culture method using the container |
| CN205856486U (en) * | 2015-12-07 | 2017-01-04 | 内江师范学院 | A kind of easy micro-fluidic chip |
| CN214735767U (en) * | 2020-11-30 | 2021-11-16 | 沈阳曼德医疗系统有限公司 | Visualization device for studying cell chemotaxis |
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2020
- 2020-11-30 CN CN202011378079.3A patent/CN112481109A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030022362A1 (en) * | 2000-11-08 | 2003-01-30 | Gregory Kirk | Cell motility and chemotaxis test device and methods of using same |
| CN103080294A (en) * | 2010-09-08 | 2013-05-01 | 株式会社岛津制作所 | Cell culture container and cell culture method using the container |
| CN205856486U (en) * | 2015-12-07 | 2017-01-04 | 内江师范学院 | A kind of easy micro-fluidic chip |
| CN214735767U (en) * | 2020-11-30 | 2021-11-16 | 沈阳曼德医疗系统有限公司 | Visualization device for studying cell chemotaxis |
Non-Patent Citations (1)
| Title |
|---|
| 宓保梅, 中国优秀硕士学位论文全文数据库/氧浓度梯度下多形核白细胞的趋化特性与机制, 15 May 2016 (2016-05-15), pages 12 - 18 * |
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